WO2013082288A1 - Glucorticoid receptor gene nr3c1 methylation in irritable bowel syndrome and other stress disorders - Google Patents

Abstract

The instant disclosure identifies sites in the glucocorticoid receptor (GR) promoter for which the degree of cytosine methylation in leukocytes can be correlated with irritable bowel syndrome (IBS) and IBS subtype. The instant disclosure also identifies GR-a and GR-β mRNA profiles in peripheral blood leukocytes that can be correlated with IBS and IBS subtype. The invention provides methods and materials that use observation of DNA methylation and/or mRNA expression to obtain information relating to irritable bowel syndrome (IBS).

Description

GLUCORTICOID RECEPTOR GENE NR3C1 METHYLATION IN IRRITABLE BOWEL SYNDROME AND OTHER STRESS DISORDERS

REFERENCE TO RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S. Provisional

Application Serial No. 61/564,761, filed November 29, 2011, the contents of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. P50

DK64539 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

This invention relates to methods of detecting and analyzing patterns of cytosine methylation and mRNA expression that are associated with pathological syndromes. 2. Description of Related Art.

Throughout development, cells and tissues differentiate and change as an organism ages. These changes include alterations to telomeres, accumulation of DNA mutations, decay of cellular and organ structures, and changes in gene expression (see, e.g. Goyns MH (2002) Mech Ageing Dev 123: 791-799). Both differentiation of tissues and aging effects are at least partially caused by chemical modifications of the genome, such as DNA methylation. In particular, the genomic DNA of higher eukaryotes contains modified nucleosides including 5-methyl cytosines. This modification is usually found as part of the dinucleotide CpG.

DNA methylation is an epigenetic determinant of gene expression. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. The consequence of methylation is usually gene silencing by prohibiting binding of DNA transcription factors. DNA methylation also correlates with other cellular processes including embryonic development, chromatin structure, genomic imprinting, somatic X-chromosome inactivation in females, inhibition of transcription and transposition of foreign DNA and timing of DNA replication. When a gene is highly methylated it is less likely to be expressed. Thus the identification of sites in the genome containing 5-meC is important in understanding cell-type specific programs of gene expression and how gene expression profiles are altered during both normal development and diseases such as cancer. Mapping of DNA methylation patterns is important for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancers.

Epigenetics is also an important research area in the understanding of complex diseases. Epigenetic changes, such as DNA methylation and histone modification, can be triggered by aging or various environmental factors implicated in the pathophysiology of several chronic diseases including cancer, chronic pain, and psychiatric diseases (see, e.g. Esteller M. N Engl J Med 2008;358: 1148-59; Denk et al, Neuron 2012;73:435-44; and Radley et al, Stress 2011;14:481-97). Epigenetic modifications can explain the variability observed in quantitative traits despite similarities in genetic background (see, e.g. Bjornsson et al, Trends Genet 2004;20:350-8).

Irritable bowel syndrome (IBS) is a symptom-based disorder without an agreed upon biomarker. In view of the inability to identify disease-specific genetic markers and the important role of environmental influences on IBS prevalence, epigenetic mechanisms have been implicated in its pathophysiology (see, e.g. Levy et al, Gastroenterology 2001;121 :799-804; Videlock et al, Gastroenterology 2009;137: 1954-62; and Dinan et al, Nat Rev Gastroenterol Hepatol 2010;7:465-71). In this context, new DNA and/or mRNA analysis techniques, for example those that can be used to characterize epigenetic and other biomarkers associated with IBS, are desirable.

SUMMARY OF THE INVENTION

As noted above, irritable bowel syndrome (IBS) is currently a symptom-based disorder. As disclosed herein, it has been discovered that methylation in the NR3C1 glucococorticoid receptor (GR) promoter and/or mRNA expression of GRa (active) and GRP (inhibitory) transcript isoforms can be correlated with a number of phenomena including the presence of irritable bowel syndrome as well as specific bowel habit subtypes associated with this pathology. These discoveries have been harnessed to provide a number of methods and materials adapted for characterizing glucococorticoid receptor (GR) promoter methylation as well as GRa and GRP transcript expression as biomarkers of IBS.

Briefly, in the experimental studies disclosed herein, NR3C1 promoter methylation was examined in peripheral blood T lymphocytes obtained from either Rome III+ IBS patients or age- and sex-matched healthy controls. In these studies, the expression of GRa (active) and GRP (inhibitory) mRNA isoforms were also measured using real-time PCR. The results of these studies showed that all but one IBS patient had methylation in the NR3C1 promoter while NR3C1 promoter methylation was not seen in any of the controls. In addition, certain GR isoform mRNA profiles were observed to correlate with IBS. Moreover, methylation patterns as well as GRP mRNA levels were further observed to differ significantly between IBS bowel habit subtypes. The data disclosed herein shows that DNA methylation in the NR3C1 promoter can be used as a biomarker for IBS as well as to characterize aspects of IBS, for example bowel habit subtypes. The data disclosed herein also shows that the expression patterns of GRa (active) and GRP (inhibitory) mRNA isoforms can also be used to characterize aspects of IBS.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, a method of examining an individual for a presence or absence of a biomarker observed in irritable bowel syndrome. In illustrative embodiments of the invention, this method comprises the steps of obtaining a biological sample derived from the individual comprising genomic DNA from leukocytes, and then observing cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1, so that the presence or absence of the biomarker observed in irritable bowel syndrome is examined. Typically, the method is performed on an individual previously selected as having one or more signs or symptoms characteristic of irritable bowel syndrome. In some embodiments of the invention, the leukocytes are obtained from the peripheral blood of the individual. In other embodiments of the invention, the leukocytes are obtained from the saliva of the individual.

Figure 4 shows the location of 8 CpG sites in the NR3C1 exon 1₇ promoter (SEQ ID NO: 1). The data presented in Figure 1 shows how GR gene methylation at these sites is increased in patients having IBS and also that specific methylation patterns are associated with IBS bowel habit subtypes. In this context, in some embodiments of the invention, cytosine methylation is observed in at least one cytosine nucleotide (and typically at least 2-6 cytosine nucleotides) selected from the group consisting of CpG site 1, 2, 3, 4, 5, 6, 7 or 8 in SEQ ID NO: 1. In some embodiments of the invention, an observation of an at least 20 % cytosine methylation at CpG site 1, 2, 3, 7 or 8 provides evidence of irritable bowel syndrome. In certain embodiments of the invention, an at least 50 % cytosine methylation at CpG site 1, 2 or 3 (and/or less than 20 % cytosine methylation at CpG site 7 or 8) provides evidence of a irritable bowel syndrome diarrhea subtype. In some embodiments of the invention, an at least 50 % cytosine methylation at CpG site 7 or 8 (and/or less than 50 % cytosine methylation at CpG site 1, 2 or 3) provides evidence of a irritable bowel syndrome constipation subtype. In some embodiments of the invention, a plurality of IBS biomarkers disclosed herein can be examined in methods of the invention. For example, in certain embodiments of invention, in addition to observing methylation patterns in the NR3C1 exon 1₇ promoter region, the method further comprises characterizing expression levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA in the leukocytes.

Related embodiments of the invention include methods of obtaining information for determining a prognosis or therapy for a patient having or suspected of having irritable bowel syndrome. These embodiments can include observing a level of glucocorticoid receptor mRNA expressed in leukocytes obtained from the patient, comparing the level of glucocorticoid receptor mRNA expressed in the leukocytes obtained from the patient with a level of glucocorticoid receptor a mRNA expressed in control leukocytes obtained from an individual not having irritable bowel syndrome, and then correlating the level of glucocorticoid receptor observed in the leukocytes obtained from the patient with a prognosis or therapy for irritable bowel syndrome. Certain embodiments of these methods further include observing a pattern of cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1.

Specific embodiments of the invention include examining the expression of glucocorticoid receptor a mRNA (SEQ ID NO: 2) and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA obtained from the leukocytes of a patient suspected of having irritable bowel syndrome. Some embodiments of the invention include correlating the level of glucocorticoid receptor mRNA observed in the leukocytes obtained from the patient with an irritable bowel syndrome subtype selected from the group consisting of irritable bowel syndrome with constipation and irritable bowel syndrome with diarrhea. In one illustrative embodiment of the invention, expression levels of glucocorticoid receptor a mRNA (SEQ ID NO: 2) in the patient's leukocytes that are at least X% below the mean levels of glucocorticoid receptor a mRNA (SEQ ID NO: 2) expressed in control leukocytes provides evidence of irritable bowel syndrome. In another illustrative embodiment of the invention, expression levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes that are at least X% above the mean levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with constipation.

As discussed in detail below, the methods of the invention can be adapted for use with a variety of art accepted processes. For example, in certain embodiments of the invention designed to examine methylation patterns in the NR3C1 exon 1₇ promoter region, a bisulfite conversion process is performed so that cytosine residues in the genomic DNA are transformed to uracil, while 5-methylcytosine residues in the genomic DNA are not transformed to uracil. Optionally, the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process. In certain embodiments of the invention, the genomic DNA is hybridized to a complimentary sequence (e.g. a synthetic polynucleotide sequence) that is coupled to a matrix (e.g. one disposed within a microarray). Optionally, the levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA are observed using a real time polymerase chain reaction process.

Embodiments of the invention also provide articles of manufacture and kits for obtaining information useful in diagnosing IBS and/or a IBS bowel habit subtype. In an illustrative embodiment, the kit includes a plurality of primers or probes specific for genomic DNA sequences in a biological sample that comprise the NR3C1 promoter region, wherein the genomic DNA sequences comprise one or more of the CG loci in the genomic DNA identified in Figure 4. Such kits of the invention can further include additional reagents, for example a reagent used in a genomic DNA polymerization process, a reagent used in a genomic DNA hybridization process, and/or a reagent used in a genomic DNA bisulfite conversion process. Optionally, the kit comprises a plurality of primer sets for amplifying mRNA and/or genomic DNA sequences.

One illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor NR3C1 promoter (SEQ ID NO: 1) and a reagent selected for use in a genomic DNA polymerization process, a genomic DNA hybridization process, or a genomic DNA bisulfite conversion process. Another illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor a mRNA (SEQ ID NO: 2) or glucocorticoid receptor β (SEQ ID NO: 3) mRNA and a reagent selected for use in a RT-PCR process (e.g. reverse transcriptase). Another illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a polynucleotide probe that hybridizes to glucocorticoid receptor a mRNA (SEQ ID NO: 2) and a polynucleotide probe that hybridizes to glucocorticoid receptor β (SEQ ID NO: 3) mRNA.

Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a graphical representation of data showing the percent DNA methylation at the eight CpG sites in the NR3C1 promoter as observed in IBS patients and for each of the IBS bowel habit subtypes. Values are the mean (± SE) percent methylation averaged for the five clones at each CpG site. Percent methylation at CpG sites 1-3 (**P=0.007) and 7-8 (**P=0.004) were significantly different among IBS bowel habit subtypes. No methylation was observed at any of the CpG sites in the control subjects.

Figure 2 provides graphical representations of data showing the mRNA expression of the active (GRa) and inhibitory (GRP) isoforms of the glucocorticoid receptor in IBS patients and healthy controls. The GRa mRNA levels (A) are significantly lower in IBS patients compared to controls (P=0.002). There was trend for a bowel habit effect on GRa mRNA expression ( =0.068). GRP mRNA levels were similar between IBS patients and controls ( =0.20), but IBS-C patients had significantly higher GRp mRNA levels compared to IBS-D and IBS-M (* *P=0.003).

Figure 3. provides a graphical representations of a predicted Cortisol response to a visceral stressor across time in IBS patients and controls from the linear mixed model is shown. This data shows that there was a significant interaction between IBS status and GRa mRNA levels with stress-induced Cortisol response ( =0.03).

Figure 4 provides a schematic showing the DNA sequence of the NR3C1 promoter area that includes 8 CpG sites examined for their methylation status.

DETAILED DESCRIPTION OF THE INVENTION

Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. All publications mentioned herein are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification. In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. All numbers recited in the specification and associated claims that refer to values that can be numerically characterized can be modified by the term "about".

The term "genome" or "genomic" as used herein is all the genetic material in the chromosomes of an organism. DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA.

The term "epigenetic" as used herein means relating to, being, or involving a modification in gene expression that is independent of DNA sequence. Epigenetic factors include modifications in gene expression that are controlled by changes in DNA methylation and chromatin structure. For example, methylation patterns are known to correlate with gene expression.

The term "nucleic acids" as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982). The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

The terms "oligonucleotide" and "polynucleotide" as used herein refers to a nucleic acid ranging from at least 2, preferable at least 8, and more preferably at least 20 nucleotides in length. Polynucleotides of the present invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced or artificially synthesized and mimetics thereof.

The term "probes" as used herein are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. The term "probe" as used herein refers to a surface-immobilized molecule that can be recognized by a particular target as well as molecules that are not immobilized and are coupled to a detectable label. The term "label" as used herein refers, for example, to colorimetric (e.g. luminescent) labels, light scattering labels or radioactive labels. Fluorescent labels include, inter alia, the commercially available fluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (ABI). See U.S. Pat. No. 6,287,778.. See U.S. Pat. No. 6,582,908 for an example of arrays having all possible combinations of probes with 10, 12, and more bases.

The term "primer" as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the complement of the 3' end of the sequence to be amplified.

The term "complementary" as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded R A or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See, M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

The term "hybridization" as used herein refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible. Factors that can affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Hybridization conditions suitable for microarrays are described in the Gene Expression Technical Manual, 2004 and the GeneChip Mapping Assay Manual, 2004, available at Affymetrix.com.

The term "array" or "microarray" as used herein refers to an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically (e.g. Illumina HumanMethylation27 microarrays). The molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

The term "biomarker" or biological marker as used herein refers to an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Illustrative biomarkers include DNA methylation profiles and mRNA expression profiles.

The term "leukocyte" as used herein refers to white blood cells that can be found, for example, in the peripheral blood and saliva and whose chief function is to protect the body against microorganisms causing disease. Illustrative leukocytes include T lymphocytes, B lymphocytes, macrophages, monocytes and the like.

Abbreviations: IBS, irritable bowel syndrome; IBS-D, IBS with diarrhea; IBS-C, IBS with constipation; IBS-M, IBS with mixed pattern; HP A, hypothalamic- pituitary adrenal; GR, glucocorticoid receptor; EAL, early adverse life events; HAD, hospital anxiety and depression scale.

DESCIPTION OF ILLUSTRATIVE ASPECTS OF THE INVENTION

Epigenomic marks can be lifelong, although potentially reversible, and can serve as the basis for sustained programming of gene expression (see, e.g. Szyf et al, Front Neuroendocrinol 2005;26: 139-62). Epigenetic programming influences gene function and phenotype, and if it affects critical genes involved in body homeostasis and physiologic functioning, it could result in chronic disease and aberrant behavior (see, e.g. Szyf et al, Biochim Biophys Acta 2009;1790:878-85). Increased stress responsiveness due to decreased negative feedback at the level of the GR associated with NR3C1 promoter methylation was demonstrated in rodents and humans (see, e.g. Coutinho et al, Am J Physiol Gastrointest Liver Physiol 2002;282:G307-G16; O'Mahony et al, Psychopharmacology (Berl) 2011;214:71-88; McGowan et al, Nat Neurosci 2009;12:342-8; Tyrka et al, PLoS One 2012;7:e30148 and Webster et al, Proc Natl Acad Sci U S A 2001;98:6865-70).

EAL refers to traumatic experiences during childhood including, but not limited to, maladjusted relationships with a parent or primary caregiver, severe illness or death of a parent, and physical, sexual or emotional abuse. EAL are more prevalent in IBS patients compared to healthy individuals and patients with organic gastrointestinal (GI) conditions (see, e.g. Bradford et al, Clin Gastroenterol Hepatol 2011 and Drossman et al., Gastroenterology 1996;111 : 1159-61). Moreover, a history of EAL increases Cortisol response to a stressful visceral stimulus (see, e.g. Videlock et al, Gastroenterology 2009;137: 1954-62). We hypothesized that stress hyperresponsiveness in IBS is associated with DNA methylation of the NR3C1 promoter, that this methylation is associated with altered GR expression, and that it may represent an epigenetic marker that differentiates IBS from healthy controls. Since DNA methylation patterns are cell and tissue specific, we measured NR3C1 gene methylation and expression in peripheral blood T cells, representing the main targets of glucocorticoid-mediated immunosuppression (see, e.g. Szyf et al, Biochim Biophys Acta 2009;1790:878-85 and Boldizsar et al, Immunobiology 2010;215:521- 6). Moreover, activated T cells are evident in IBS (see, e.g. Ohman et al., Am J Gastroenterol 2009;104: 1205-12).

Early life stress, as well as chronic or severe stress in predisposed adults, can result in enhanced responsiveness of central stress circuits, dysregulation of negative feedback regulation of the stress response, and increased vulnerability to chronic visceral pain disorders, including IBS (see, e.g. Chang et al, Gastroenterology 2011;140:761-5). For example, maternal separation, an accepted model of EAL, predisposed adult rats to develop stress-induced visceral hypersensitivity and defecation, increased intestinal permeability and corticosterone levels, and anxiety- like behavior, similar to findings reported in IBS (see, e.g. Coutinho et al, Am J Physiol Gastrointest Liver Physiol 2002;282:G307-G16; Gareau et al, Am J Physiol Gastrointest Liver Physiol 2007;293:G198-203 and Gareau et al., Pediatr Res 2006;59:83-8). Decreased GR mRNA in the frontal cortex and increased corticotropin releasing factor (CRF) mRNA density in the paraventricular nucleus and amygdala were reported in animals exposed to maternal separation (see, e.g. Ladd et al, Psychoneuroendocrinology 2005;30:520-33). Blockade of GRs in the amygdala abolished stress-induced visceral hyperalgesia (see, e.g. Myers et al, Am J Physiol Gastrointest Liver Physiol 2012;302:G260-6). Additionally, elevated corticosterone levels in rodents increased responsiveness of the amygdala, related central arousal circuits, and colonic sensitivity via CRF (see, e.g. Myers et al, Behav Brain Res 2005;161 :39-44 and Johnson et al, PLoS One 2010;5:e8573). Perinatal maternal care is also linked to epigenetic alterations at the NR3C1 gene in the adult offspring (see, e.g. O'Mahony et al., Psychopharmacology (Berl) 2011;214:71-88 and Coutinho et al., Gastroenterology 2000; 118:A637). The associated molecular signatures include increased methylation of the NR3C1 exon 1₇ promoter, decreased hippocampal GR expression, increased hypothalamic CRF mRNA expression and basal and stress- induced adrenocorticotropin hormone (ACTH) and corticosterone levels (see, e.g. Liu et al, Science 1997;277: 1659-62 and Meaney et al, Trends Mol Med 2007;13:269- 77).

Consistent with the experimental data disclosed herein, other studies also support a similar relationship of EAL and increased NR3C1 promoter methylation (see, e.g. McGowan et al, Nat Neurosci 2009;12:342-8; Oberlander et al, Epigenetics 2008;3:97-106 and Tyrka et al, PLoS One 2012;7:e30148). CpG sites 1-8 corresponded to sites 31-38 in a study where increased methylation was found in abused vs. non-abused suicides and controls at CpG sites corresponding to our sites 1- 2 (see, e.g. McGowan et al, Nat Neurosci 2009;12:342-8). In line with the data presented herein, little (<10%) or no methylation was seen in controls. Our CpG sites 5-8 corresponded to sites 1-4 in a study in which increased methylation of CpG 3 from cord blood leukocytes correlated with prenatal maternal depression scores (a form of EAL) and higher stress-induced Cortisol response in newborns (see, e.g. Oberlander et al, Epigenetics 2008;3:97-106). In one study, mean % methylation at CpG sites corresponding to our sites 5 and 7 was <10% in healthy volunteers and modestly correlated with lack of adequate nurturing during childhood (see, e.g. Tyrka et al, PLoS One 2012;7:e30148). However, since EAL was also reported by almost half of our controls, it does not explain the striking differences between IBS and controls. Epigenetic programming of the NR3C1 gene can be influenced in utero and early after birth by poor nutrition, methyl-deficiency, glucocorticoid elevations to stress, and other chemical factors. After birth, social environmental exposures can elicit central neuronal signaling pathways that in turn signal modifications in methylation (see, e.g. Szyf et al, Biochim Biophys Acta 2009;1790:878-85). Our eight CpG sites in the NR3C1 promoter were located within the exon IF, the human homologue of the rat exon l_7j representing putative binding sites of the transcription factor nerve growth factor-inducible protein A (NGFI-A) (see, e.g. McGowan et al, Nat Neurosci 2009;12:342-8).

Hypothesizing that increased methylation of the NR3C1 promoter would be associated with decreased expression of the GR, we measured expression of active (GRa) and inhibitory (GRP) isoforms, generated by alternative splicing of the primary transcript (see, e.g. Oakley et al, J Biol Chem 2011;286:3177-84). GRa mRNA expression was significantly lower in IBS patients compared to controls. GRa binds glucocorticoids and activates the transcription of hormone-sensitive genes (see, e.g. Webster et al, Proc Natl Acad Sci U S A 2001;98:6865-70). In contrast to our findings in IBS patients, levels of GRa and GRP mRNA in peripheral blood mononuclear cells were similar between patients with active and inactive inflammatory bowel disease and healthy controls, with the exception of higher GRP levels in active ulcerative colitis (see, e.g. Orii et al., Biochem Biophys Res Comrnun 2002;296: 1286-94).

Changes in GR mRNA blood levels are similar to that in the hippocampus following chronic administration of glucocorticoids (see, e.g. Lee et al, Endocrinology 2010;151 :4332-43). Lower GRa expression would be expected to be associated with an enhanced Cortisol response in IBS due to decreased negative feedback of Cortisol via GRs. Consistent with this hypothesis, we found that IBS patients with lower expression of GRa had a greater Cortisol response to a stressful visceral stimulus than patients with higher expression, suggesting that hyperreactivity of the HPA axis is due to decreased negative feedback of Cortisol. In contrast, healthy controls had no NR3C1 promoter methylation and higher GRa mRNA levels. Healthy subjects who had higher Cortisol responses to an acute stressor also had a correspondingly greater expression of GRa (see, e.g. Alderman et al, Gen Comp Endocrinol 2012;176:79-85). Studies in healthy volunteers found modest correlations between NR3C1 promoter methylation and Cortisol responses but neither measured GR mRNA levels (see, e.g. Tyrka et al, PLoS One 2012;7:e30148 and de Rooij et al, Psychoneuroendocrinology 2011). The lack of differences in Cortisol response between IBS and controls is similar to our previous study with a different group of subjects (see, e.g. Videlock et al, Gastroenterology 2009;137: 1954-62). Thus, Cortisol response may be affected differently by GRa mRNA levels in IBS patients and controls.

As discussed in detail below, NR3C1 promoter methylation and GR mRNA expression differed between IBS bowel habit subtypes. The lower expression of GRa and higher expression of GRP provides evidence that increased methylation at CpG sites 7-8 in the NR3C1 promoter is more influential in GR transcription. In fact, GRP mRNA levels positively correlated with % methylation at CpG sites 7-8. GRP inhibits GRa transcription and can act as a negative inhibitor of GRa on glucocorticoid- regulated genes. GRP can also directly induce and repress genes not controlled by GRa (see, e.g. Oakley et al, J Biol Chem 2011;286:3177-84). Thus, GRp may induce methylation of the NR3C1 promoter. GRs have a broad range of physiologic effects and may affect central (e.g., amygdala) and/or peripheral (e.g., enteric nervous system) mechanisms involved in peristalsis or secretion.

In summary, NR3C1 promoter methylation is shown to differentiate IBS patients from controls, is associated with decreased GRa expression, and may explain the well documented propensity of IBS patients to stress-sensitivity of their symptoms (see, e.g. Mayer et al, Am J Physiol Gastrointest Liver Physiol 2001;280:G519-G24). IBS bowel habit subtypes are also observed to differ in their NR3C1 methylation pattern and expression of active and inhibitory isoforms of GR. Specifically, as disclosed herein, IBS patients compared to healthy control subjects showed DNA methylation of the NR3C1 promoter and lower GRa mRNA expression. In addition, bowel habit predominance was associated with distinct differences in the methylation pattern of the NR3C1 promoter and expression of GR. Finally, decreased GR expression was associated with a greater Cortisol response to a stressful visceral stimulus in patients, but not healthy controls.

In this context, embodiments of the invention include, for example, methods of examining an individual for a presence or absence of a biomarker observed in irritable bowel syndrome and/or one specifically associated with an IBS bowel habit subtype. In illustrative embodiments of the invention, such methods comprise the steps of obtaining a biological sample derived from the individual comprising genomic DNA from leukocytes, and then observing cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1, so that the presence or absence of the biomarker observed in irritable bowel syndrome is examined. In some embodiments of the invention, the leukocytes are obtained from the peripheral blood of the patient. In other embodiments of the invention, the leukocytes are obtained from the saliva of the patient. In certain embodiments of the invention, the genomic DNA of epithelial cells is observed instead of, or in addition to, the genomic DNA of leukocytes.

Figure 4 shows the sequence and location of 8 CpG sites in the NR3C1 exon 1₇ promoter (SEQ ID NO: 1). Figure 1 shows that GR gene methylation at these 8 CpG sites is increased in IBS. Figure 1 also shows IBS methylation patterns that are bowel habit specific. The methylation patterns disclosed in Figure 1 allow those of skill in the art to harness these patterns for diagnostic tests that, for example, examine the methylation of one or more of the 8 CpG sites shown in SEQ ID NO: 1 as a biomarker for IBS and/or an IBS bowel habit subtype. In some embodiments of the invention, cytosine methylation is observed in at least one cytosine nucleotide (and typically at least 2, 3, 4, 5, or 6 cytosine nucleotides) selected from the group consisting of CpG site 1, 2, 3, 4, 5, 6, 7 or 8 in SEQ ID NO: 1. In some embodiments of the invention, an observation of an at least 20, 30, 40, 50, 60 or 70 % cytosine methylation at CpG site 1, 2, 3, 7 or 8 provides evidence of irritable bowel syndrome (see, e.g. Figure 1). In certain embodiments of the invention, an at least 50, 60, 70 or 80 % cytosine methylation at CpG site 1, 2 or 3 (and/or less than 40, 30 or 20 % cytosine methylation at CpG site 7 or 8) provides evidence of a irritable bowel syndrome diarrhea subtype (see, e.g. Figure 1). In some embodiments of the invention, an at least 50, 60 or 70 % cytosine methylation at CpG site 7 or 8 (and/or less than 50 or 40 % cytosine methylation at CpG site 1, 2 or 3) provides evidence of a irritable bowel syndrome constipation subtype (see, e.g. Figure 1). In certain embodiments of the invention, a between 20% and 40 % cytosine methylation at CpG site 1, 2, 3, 7 and 8) provides evidence of a irritable bowel syndrome mixed pattern subtype (see, e.g. Figure 1).

A plurality of the IBS biomarkers disclosed herein can be examined in methods of the invention. For example, in certain embodiments of invention, in addition to observing methylation patterns in the NR3C1 exon 1₇ promoter region, the method further comprises characterizing expression levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA in leukocytes. In an illustrative embodiment of the invention DNA samples (e.g. 1 μg genomic DAN obtained from a peripheral blood monocyte) can be bisulfite-treated and used for PCR analysis covering the NR3C1 promoter area. A total number of clones can sequenced to measure methylation status at each of the 8 CpG sites of the NR3C1 promoter. Percent methylation can then be calculated as: number of clones with methylation/total number x 100.

Embodiments of the invention include methods of obtaining information for determining a prognosis or therapy for a patient having or suspected of having irritable bowel syndrome. As noted above, certain embodiments of these methods for observing the include observing a pattern of cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1. Other embodiments include observing a level of glucocorticoid receptor mRNA expressed in leukocytes obtained from the patient, comparing the level of glucocorticoid receptor mRNA expressed in the leukocytes obtained from the patient with a level of glucocorticoid receptor a mRNA expressed in control leukocytes obtained from an individual not having irritable bowel syndrome, and then correlating the level of glucocorticoid receptor observed in the leukocytes obtained from the patient with a prognosis or therapy for irritable bowel syndrome. Certain embodiments of these methods for observing the levels of glucocorticoid receptor mRNAs further include observing a pattern of cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1. In some embodiments of the invention, additional methodological analyses are performed in addition to these methylation or mRNA analyses, for example those that comprise the measurement Cortisol and/or progesterone levels.

Typically, methylation patterns and/or GRa and GRP mRNA profiles are examined in individuals identified as having one or more signs or symptoms characteristic of irritable bowel syndrome. As is known in the art, signs or symptoms characteristic of irritable bowel syndrome can include abdominal pain, fullness, gas, and bloating that have been present for at least 3 days a month for the last 3 months. Because the methylation patterns and/or GRa and GRP mRNA profiles disclosed herein are shown to be specific for IBS, certain embodiments of the invention are used to diagnose IBS (or alternatively to rule out IBS) in a patient having one or more signs or symptoms found in both IBS as well as other syndromes such as inflammatory bowel disease (IBD), celiac disease and colon cancer.

In some embodiments of the invention, the expression profile of glucocorticoid receptor a mRNA (SEQ ID NO: 2) and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA is characterized. Such embodiments of the invention can include the step of correlating the level of glucocorticoid receptor mRNA observed in leukocytes obtained from the patient with a irritable bowel syndrome subtype selected from the group consisting of irritable bowel syndrome with constipation and irritable bowel syndrome with diarrhea. In one illustrative embodiment of the invention, expression levels of glucocorticoid receptor a mRNA (SEQ ID NO: 2) in the patient's leukocytes that are below at least the 10^th percentile distribution of glucocorticoid receptor a mRNA (SEQ ID NO: 2) expressed in control leukocytes (and/or predetermined mRNA expression levels observed in leukocytes from individuals who do not have IBS) provides evidence of irritable bowel syndrome. In another illustrative embodiment of the invention, expression levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes that are at least 10% above the mean levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with constipation. In addition, expression levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes that are below at least 10% below the mean levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with diarrhea. Expression levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes can distinguish the constipation and diarrhea subtypes of irritable bowel syndrome. Expression levels of the glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes that are below the 25^th percentile of the distribution of the levels of the glucocorticoid receptor β mRNA (SEQ ID NO: 3) expressed in the leukocytes from irritable bowel syndrome with constipation provides evidence of irritable bowel syndrome with diarrhea.

Embodiments of the invention include methods that comprise examining individuals for the presence or absence of one or more biomarkers observed in irritable bowel syndrome (e.g. so as to obtain information for determining a prognosis or therapy for a patient having or suspected of having irritable bowel syndrome); and then administering a therapeutic agent to those individuals identified as expressing such biomarkers. Therapeutic agents used to treat IBS include those designed to treat diarrhea such as diphenoxylate (e.g. Lomotil), loperamide (e.g. Imodium), antibiotics (e.g. Xifaxin) and Alosetron (e.g. Lotronex). Therapeutic agents used to treat IBS also include those designed to treat constipation such as Lubiprostone (e.g. Amitiza), Linaclotide (e.g. Linzess), osmotic laxatives (e.g. Milk of Magnesia and nonabsorbable sugars such as lactulose), and polyethylene glycol compositions (e.g. MiraLax). Therapeutic agents used to treat IBS also include tricyclic antidepressants (e.g., Elavil), selective serotonin reuptake inhibitors (e.g. Celexa), serotonin- norepinephrine reuptake inhibitors (e.g., Cymbalta), and bile acid binding agents such as cholestyramine (e.g. Questran). Non-pharmacologic treatments have also been used to treat IBS include cognitive behavioral therapy, stress management, mindfulness meditation, psychotherapy, yoga, acupuncture. In some embodiments of the invention, the therapeutic agent is selected following the observation of an IBS biomarker profile that is characteristic of a specific bowel habit subtype. For example, in one such embodiment of the invention, a patient identified as having Irritable Bowel Syndrome with a constipation subtype (IBS-C) is administered Lubiprostone. In another example of an embodiment of the invention, the IBS biomarker profile can predict treatment response. For example, a therapeutic regimen is selected following the observation of the IBS biomarker profile that is associated with a symptomatic response to this treatment.

As discussed in detail below, the methods of the invention can be adapted for use with a variety of art accepted processes. For example, in certain embodiments of the invention designed to observe methylation patterns, a bisulfite conversion process is performed so that cytosine residues in the genomic DNA are transformed to uracil, while 5-methylcytosine residues in the genomic DNA are not transformed to uracil. Optionally, the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process. In certain embodiments of the invention, the genomic DNA is hybridized to a complimentary sequence (e.g. a synthetic polynucleotide sequence) that is coupled to a matrix (e.g. one disposed within a microarray). Optionally in embodiments of the invention that observe GR mRNA levels, the levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA are observed using a real time polymerase chain reaction process.

Embodiments of the invention also provide articles of manufacture and kits for obtaining information useful to determine the age of an individual. In an illustrative embodiment, the kit includes a plurality of primers or probes specific for at least two genomic DNA sequences in a biological sample, wherein the genomic DNA sequences comprise one or more of the CG loci in the genomic DNA identified in Table 3 and Figure 4. Such kits of the invention can further include additional reagents, for example a reagent used in a genomic DNA polymerization process, a reagent used in a genomic DNA hybridization process, and/or a reagent used in a genomic DNA bisulfite conversion process (e.g. a buffering composition comprising a surfactant such a TRITON or TWEEN surfactant). Optionally, the kit comprises a plurality of primer sets for amplifying mRNA and/or genomic DNA sequences.

One illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor NR3C1 promoter (SEQ ID NO: 1) and a reagent selected for use in a genomic DNA polymerization process (e.g. a composition comprising a mixture of dNTPs), a genomic DNA hybridization process, or a genomic DNA bisulfite conversion process (e.g. a sodium metabisulfate composition). Another illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor a mRNA (SEQ ID NO: 2) or glucocorticoid receptor β (SEQ ID NO: 3) mRNA and a reagent selected for use in a RT-PCR process (e.g. a reverse transcriptase). Another illustrative embodiment of the invention is a kit for examining a biomarker observed in irritable bowel syndrome that comprises a polynucleotide probe that hybridizes to glucocorticoid receptor a mRNA (SEQ ID NO: 2) and a polynucleotide probe that hybridizes to glucocorticoid receptor β (SEQ ID NO: 3) mRNA.

As noted above, embodiments of the invention are designed to detect methylation status or patterns in SEQ ID NO: 1. A variety of methods for detecting methylation status or patterns have been described in, for example U.S. Pat. Nos. 6,214,556, 5,786,146, 6,017,704, 6,265,171, 6,200,756, 6,251,594, 5,912,147, 6,331,393, 6,605,432, and 6,300,071 and US Patent Application publication Nos. 20030148327, 20030148326, 20030143606, 20030082609 and 20050009059, each of which are incorporated herein by reference. Other array based methods of methylation analysis are disclosed in U.S. patent application Ser. No. 11/058,566. For a review of some methylation detection methods, see, Oakeley, E. J., Pharmacology & Therapeutics 84:389-400 (1999). Available methods include, but are not limited to: reverse-phase HPLC, thin-layer chromatography, Sssl methyltransferases with incorporation of labeled methyl groups, the chloracetaldehyde reaction, differentially sensitive restriction enzymes, hydrazine or permanganate treatment (m5C is cleaved by permanganate treatment but not by hydrazine treatment), sodium bisulfite, combined bisulphate-restriction analysis, and methylation sensitive single nucleotide primer extension.

Methods of measuring DNA methylation include those that allows the quantitative analysis of a single cytosine using SYBR green-based Real-time PCR. Briefly, following bilsulfite conversion, the sequence of interest is amplified by PCR. Then, using primer designed specifically to prime the modified nucleotide, the sequence of interest is quantified by Real-time PCR using SYBR green. The results are normalized by a real-time PCR reaction that is independent of methylation. Such techniques are described, for example in Dugast-Darzacq et al., Methods Mol Biol. 2009;507:281-303. PubMed PMID: 18987822; Chan et al, Biotechnol Lett. 2004 Aug;26(16): 1289-93. PubMed PMID: 15483389; Gustafson J Mol Diagn. 2008 Jan;10(l):33-42. Epub 2007 Dec 28. PubMed PMID: 18165272; PubMed Central PMCID PMC2175541.

Embodiments of the invention include a variety of art accepted technical processes. For example, in certain embodiments of the invention, a bisulfite conversion process is performed so that cytosine residues in the genomic DNA are transformed to uracil, while 5-methylcytosine residues in the genomic DNA are not transformed to uracil. Kits for DNA bisulfite modification are commercially available from, for example, Human Genetic Signatures' Methyleasy and Chemicon's CpGenome Modification Kit. See also, WO04096825A1, which describes bisulfite modification methods and Olek et al. Nuc. Acids Res. 24:5064-6 (1994), which discloses methods of performing bisulfite treatment and subsequent amplification. Bisulfite treatment allows the methylation status of cytosines to be detected by a variety of methods. For example, any method that may be used to detect a SNP may be used, for examples, see Syvanen, Nature Rev. Gen. 2:930-942 (2001). Methods such as single base extension (SBE) may be used or hybridization of sequence specific probes similar to allele specific hybridization methods. In another aspect the Molecular Inversion Probe (MIP) assay may be used.

In certain embodiment of the invention, the genomic DNA is hybridized to a complimentary sequence (e.g. a synthetic polynucleotide sequence) that is coupled to a matrix (e.g. one disposed within a microarray). Optionally, the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process. For example, prior to or concurrent with hybridization to an array, the sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, for example, PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al, Academic Press, San Diego, Calif, 1990); Mattila et al, Nucleic Acids Res. 19, 4967 (1991); Eckert et al, PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al, IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070 which is incorporated herein by reference. EXAMPLES

The present invention is described in detail in the following example, but is not limited by any aspect of this example.

EXAMPLE 1: EXPERIMENTAL PROTOCOLS SHOWING TYPICAL

METHODS AND MATERIALS USEFUL FOR PRACTICING EMBODIMENTS OF THE INVENTION

METHODS

Study subjects IBS patients and healthy individuals between the ages of 18 and 55 were recruited mainly from community advertisements although subjects were not consecutively enrolled. All IBS patients met the Rome III diagnostic criteria for IBS. Bowel habit subtypes of IBS with diarrhea (IBS-D), constipation (IBS-C) and mixed pattern (IBS-M) were based on the Rome III criteria (see, e.g. Longstreth et al., Gastroenterology 2006;130 1480-91). Control subjects did not have IBS, other chronic pain conditions, infectious or inflammatory disorders, or current history of psychiatric illness, and were not taking centrally acting drugs. None of the subjects had taken corticosteroids in the past one year or had a current history of tobacco or alcohol abuse.

Symptom Measures

IBS symptoms were assessed using a bowel symptom questionnaire, which included a numeric rating scale for IBS symptoms over the past week. Anxiety and depression symptoms were measured with the Hospital Anxiety and Depression (HAD) scale (see, e.g. Zigmond et al, Acta Psychiatr Scand 1983;67:361-70). A history of psychiatric disorders was evaluated using a structured clinical interview for the DSM-IV (MINI) (see, e.g. Sheehan et al, J Clin Psychiatry 1998;59 Suppl 20:22- 33;quiz 4-57). A history of trauma or abuse was assessed by the Trauma History Questionnaire (see, e.g. Green B. The Trauma History Questionnaire. Lutherville, MD: Sidran Press; 1996). "Early life" was defined as under the age of 18.

Study Protocol

Similar to a previous study, a flexible sigmoidoscopy to at least 40 cm from the anal verge was performed between 12:00pm-2:00pm (see, e.g. Videlock et al, Gastroenterology 2009;137: 1954-62). Prior to the sigmoidoscopy, blood samples were drawn for isolation of T lymphocytes. Saliva was collected at 7 time -points for Cortisol levels: baseline, immediately following sigmoidoscopy, and 10, 20, 30, 45, and 60 minutes after sigmoidoscopy (see, e.g. Videlock et al, Gastroenterology 2009;137: 1954-62). Saliva for progesterone was obtained to confirm menstrual cycle phase.

DNA and RNA extraction from peripheral blood T lymphocytes

Twenty mL blood was drawn into heparinized tubes and was processed in the

Clinical Immunology Research Laboratory at UCLA. The blood was centrifuged at 1500 RPM for 10 min and the plasma was removed. Dulbecco's Phosphate Buffered Saline lx (DPBS) (Sigma- Aldrich Co. LLC, St. Louis, MO) was added in a 1 : 1 ratio to the remaining blood cells. For T lymphocyte isolation, 3-4 mL of Histopaque^®- 1077 Hybri-Max^™ (Sigma- Aldrich Co. LLC, St. Louis, MO) was added into the cell mixture and centrifuged for 20 min at 2000 RPM. After removing DPBS/plasma, Hanks' Balanced Salt solution (Sigma-Aldrich Co. LLC, St. Louis, MO) was added to the remaining blood cells (-0.5 mL) so that the final volume was 12 mL. The cell mixture was centrifuged for 10 min at 1500 RPM. One mL of 20% Human Serum AB, which was prepared by mixing 80% RPMI 1640 Media (Sigma-Aldrich Co. LLC, St. Louis, MO) and 20% Human AB serum (Gemini Bio-products, West Sacramento, CA) was added. Cell count and viability of at least 20 million cells were determined using the Coulter AC-T Diff Hematology Analyzer (Beckman Coulter, Inc., Brea, CA) and DHC-N01 Neubauer hemacytometer (Incyto, Korea). Before storing, 20% Dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co. LLC, St. Louis, MO) was added. The samples were stored in a liquid nitrogen freezer.

The UCLA Clinical Microarray Core isolated DNA and RNA from T lymphocytes by first processing the cells using the MagNA Pure Compact Nucleid Acid Isolation Kit I (Roche Applied Science, Indianapolis, IN) and MagNA Pure Compact RNA Isolation Kit (Roche Applied Science, Indianapolis, IN), respectively, adhering to the kits' protocol. They isolated DNA and RNA using MagNA Pure Compact Instrument (Roche Applied Science, Indianapolis, IN).

NR3C1 promoter methylation and mRNA expression Briefly, genomic DNA samples (1 μg) were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen, Germantown, MD). A total of five clones per sample were sequenced to assess methylation status of CpG sites in the NR3C1 promoter (Figure 4). Real-time RT-PCR was performed to determine the expression levels of GRa and GRp. Reverse Transcription was carried out using the RETROscript Kit (Ambion Inc., Austin, TX). GR is used to refer to mRNA expression of these isoforms and NR3C1 to refer to promoter methylation of GR (see, e.g. McGowan et al., Nat Neurosci 2009;12:342-8 and Oakley et al, J Biol Chem 2011;286:3177-84).

Genomic DNA samples (1 μg) were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen, Germantown, MD) following the manufacturer's suggested protocol, and stored at -20°C until further analysis. The bisulfite-treated DNA was used for PCR analysis (forward primer: 5'GGGTTCTGCTTTG CAACTTCgccactcacgcagctcag-3 ' (SEQ ID NO: 4) and reverse primer: 5 '-CCTTTTTCCTGGGGAGTTG gttaagagggccaccgagtt-3 ' (SEQ ID NO: 5)) covering the NR3C1 promoter area (536195-bp) (Genebank accession number AY436590). The PCR product was analyzed and extracted from the agarose gel using Qiaquick Gel Extraction kit (Qiagen gel extraction kit) and cloned into TOPO-TA vector (TOPO-TA cloning kit, Invitrogen). A total of 5 clones per sample were sent for sequencing. Sequencing will reveal the methylation status of the CpG sites in the promoter area of NR3C1.

Salivary Cortisol and progesterone measurements

Salivary Cortisol and progesterone levels were measured using the Salivary Cortisol Enzyme Immunoassay Kit (Salimetrics, LLC, State College, PA) and Microplate Readers (Bio-Rad Laboratories, Inc., Hercules, CA). Intra- and inter- assay variation for Cortisol was less than 5% and 7%, respectively. Intra-and inter- assay variation for progesterone was less than 6.2% and 7.5%, respectively.

Statistical analysis Descriptive statistics of baseline measures were conducted for categorical (frequency [%]) and continuous (mean±SE) variables. Kruskal-Wallis and Fisher's Exact tests were utilized to test group differences among continuous variables or categorical variables respectively. Spearman's correlation was used to assess the linear association between continuous variables. Cortisol responses were evaluated by a linear mixed model regression analysis with the Cortisol response as the within- subject factor and IBS status as the between- subject factor. Interaction between the GRa mRNA with IBS status was tested in the linear mixed models. The best-fit covariance structure was determined by likelihood ratio tests and the one yielding a low Akaike's Information Criteria (AIC). Any non-normally distributed variables were log-transformed for the regression analysis. Significance was assessed at a 0.05 level. All analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA) or R version 2.14.0. RESULTS

Clinical characteristics

The clinical characteristics of the 23 IBS patients and 23 control subjects were similar (Table 1). Even though within the normal range, anxiety symptom scores were higher in IBS patients vs. controls (P=0.03). Two controls and six IBS patients had a history of current or past psychiatric disorder. Only one subject (IBS patient) was taking antidepressants (bupropion and sertraline). Sigmoidoscopies were normal in all subjects.

NR3C1 (GR) DNA methylation

DNA methylation status of eight CpG sites in the NR3C1 promoter are shown in Figure 4. In controls, NR3C1 methylation was not present at any of the CpG sites. In contrast, all but one IBS patient had methylation of the NR3C1 promoter (Table 2). The IBS patient without methylation was a women with IBS-M with no history of EAL or psychiatric disease. Methylation patterns by IBS bowel habit subtypes across the eight CpG sites clustered into three distinct groups, where % methylation was higher at CpG sites 1-3 in IBS-D, but higher at CpG sites 7-8 in IBS-C patients (Figure 1). Since percent methylation was low at CpG sites 4-6 for most IBS patients, we averaged % methylation within CpG sites 1-3 and 7-8 when comparing across IBS bowel habit subtypes. Percent methylation at CpG sites 1-3 (P=0.007) and 7-8 (P=0.004) were significantly different among IBS bowel habit subtypes. The degree of methylation at CpG sites 1-3 positively correlated with depression symptom scores (r=0.52, =0.01), but there was no association with sex or IBS symptom severity. NR3C1 mRNA expression

We measured mRNA expression of both the active (GRa) and inhibitory (GRP) iso forms of the NR3C1 in peripheral blood lymphocytes. GRa mRNA levels were significantly lower in IBS patients vs. controls (P=0.002, Figure 2A). Interestingly, the lower values in IBS vs. controls were mainly seen in men (0.56+0.06 vs. 1.01+0.05, =0.003), but not women (0.75+0.07 vs. 0.88+0.04, =0.192). There was a trend for a bowel habit effect on GRa mRNA expression (IBS- C: 0.44+0.09, IBS-D: 0.69+0.12, IBS-M: 0.85+0.09, =0.068, Figure 2A). GRp mRNA levels were not significantly different between IBS patients and controls ( =0.20, Figure 2B). However, IBS-C patients had significantly higher GRP mRNA levels compared to IBS-D and IBS-M ( =0.003, Figure 2B). There was a strong positive correlation between GRP mRNA levels and % methylation at CpG sites 7-8 (r=0.682, =0.006). GRa mRNA levels did not correlate with % methylation.

Real time PCR analysis

Real-time RT-PCR was performed to determine the expression levels of GRa and GRp. Reverse Transcription was carried out using the RETROscript Kit (Ambion Inc., Austin, TX). Real-time PCR was carried out in triplicate using the iQ SYBR green supermix (Bio-Rad) on a CFX384 Real-Time System (Bio-Rad). Cycling conditions were as follows: 95°C for 3 minutes followed by 40 amplification cycles (95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds). GRa and GRp expression levels were normalized to the levels of β-Actin and GAPDH. Primers used for GRa, forward: 5 '-CCATTGTCAAG AGGGAAGGAA-3 ' (SEQ ID NO: 6) and reverse: 5 ' -TGTTTGGAAGC AATA GTTAAGGAG-3 ' (SEQ ID NO: 7); for GRp, forward: 5 '-AGAACTGGCAGCGGTTTTATC-3 ' (SEQ ID NO: 8) and reverse: 5'- GTGTGAGATGTGCTTTCTGGTTT-3 ' (SEQ ID NO: 9); for β-Actin, forward: 5'- CCCA GCAC AATGAAGATC AA-3 ' (SEQ ID NO: 10) and reverse: 5'- ACATCTGCTGGAAGGTGGAC-3 ' (SEQ ID NO: 11); for GAPDH, forward: 5'- ATGTTCGTCATGGGTGTGAA-3 ' (SEQ ID NO: 12) and reverse: 5'- GGTGCTAAGCAGTTGGTGGT-3 ' (SEQ ID NO: 13).

Stress-induced Cortisol response

Cortisol levels significantly increased following sigmoidoscopy ( =0.002), but there was no significant difference between IBS patients and controls. There was a significant interaction between IBS status and GRa mRNA levels with stress-induced Cortisol response (P=0.03, Figure 3). In controls, higher Cortisol levels were associated with higher GRa mRNA expression. In contrast, IBS patients with lower GRa mRNA expression had a greater Cortisol response than those with higher expression.

Effect of EAL

Within IBS patients, a history of EAL was associated with higher % NR3C1 promoter methylation at CpG sites 1-3 compared to those without EAL (80% vs. 41.5%, =0.028). Among all subjects, those with a history of EAL demonstrated a significant increase in Cortisol levels following sigmoidoscopy, compared to subjects without EAL ( =0.02). EAL was not associated with GRa or GR mRNA expression.

TABLES Table 1. Clinical characteristics of study subjects

Abbreviations: BMI- body mass index; IBS-C- IBS with constipation; IBS-D- IBS with diarrhea; IBS-M- IBS with mixed pattern; HAD- Hospital Anxiety and Depression Scale; EAL- early adverse life events.

^XEAL history was obtained in 20 IBS patients and 18 healthy controls.

Past history of Axis 1 disorder in controls and IBS patients: major depressive disorder. One IBS patient also had a past history of alcohol abuse.

Current history of Axis disorder in IBS patients: major depressive disorder (n=l) and generalized anxiety disorder (n=2, one had coexistent obsessive-compulsive disorder and social phobia and one had a past history of alcohol abuse).

2For premenopausal women not taking oral contraceptive agents or provera, menstrual cycle phase was determined by the count forward/backward method (menses: first three days of menses; follicular: days 4-14; luteal: day 14 - onset of menses). Salivary progesterone was collected to help confirm cycle phase.

Table 2. Methylation Status of NR3C1 (GR) Promoter

Mean ± SE % methylation of NR3C1 from DNA extracted from T-cells in the blood in IBS patients and healthy controls. Note that controls have no methylation. Percent methylation is significantly different at CpG sites 1-3 (** =0.007) and 7-8 (** =0.004) among the three IBS bowel habit subtypes.

Abbreviations: IBS-C- IBS with constipation; IBS-D- IBS with diarrhea; IBS-M- IBS with mixed pattern

TABLE 3: GR POLYNUCLEOTIDE SEQUENCES

A. NR3C1 PROMOTER SEQUENCE:

GCGCTTGCCGCCAAGGGGCAGAGCGAGCTCCCGAGTGGGTCTGGAGCCGC GGAGCTGGGCGGGGGCGGGA (SEQ ID NO : 1 )

See also Genbank accession number AY436590.

B. SEQ ID NO: 1 showing CpG sites 1-8 capitalized in boldface type: gCGcttgcCGccaaggggcagagCGagctccCGagtgggtctggagcCGCGgagctgggCGggggC

Ggga

As shown in Figure 4:

CpG site 1 corresponds to cytosine position number 2 in SEQ ID NO: 1.

CpG site 2 corresponds to cytosine position number 9 in SEQ ID NO: 1.

CpG site 3 corresponds to cytosine position number 24 in SEQ ID NO

CpG site 4 corresponds to cytosine position number 32 in SEQ ID NO

CpG site 5 corresponds to cytosine position number 48 in SEQ ID NO

CpG site 6 corresponds to cytosine position number 50 in SEQ ID NO

CpG site 7 corresponds to cytosine position number 60 in SEQ ID NO

CpG site 8 corresponds to cytosine position number 66 in SEQ ID NO

C. GLUCOCORTICOID RECEPTOR a Real time PCR product (119bp):

CCATTGTCAAGAGGGAAGGAAactccagccagaactggcagcggttttat

caactgacaaaactcttggattctatgcatgaagtggttgaaaatCTCCT TAACTATTGCTTCCAAACA (SEQ ID NO: 14)

Genbank Accession Numbers for GRa are (transcript variants 1,2,3,4,5,7): NM_001204264 (for variant 1 )

NM_001018074 (for variant 2)

NM_001018075 (for variant 3)

NM 001018076 (for variant 4)

NM_001018077 (for variant 5)

NM_001204261 (for variant 7)

GGCGCCGCCTCCACCCGCTCCCCGCTCGGTCCCGCTCGCTCGCCCAGGCCGGGCTGC CCTTTCGCGTGTCCGCGCTCTCTTCCCTCCGCCGCCGCCTCCTCCATTTTGCGAGCT CGTGTCTGTGACGGGAGCCCGAGTCACCGCCTGCCCGTCGGGGACGGATTCTGTGGG TGGAAGGAGACGCCGCAGCCGGAGCGGCCGAAGCAGCTGGGACCGGGACGGGGCACG CGCGCCCGGAACCTCGACCCGCGGAGCCCGGCGCGGGGCGGAGGGCTGGCTTGTCAG CTGGGCAATGGGAGACTTTCTTAAATAGGGGCTCTCCCCCCACCCATGGAGAAAGGG GCGGCTGTTTACTTCCTTTTTTTAGAAAAAAAAAATATATTTCCCTCCTGCTCCTTC TGCGTTCACAAGCTAAGTTGTTTATCTCGGCTGCGGCGGGAACTGCGGACGGTGGCG GGCGAGCGGCTCCTCTGCCAGAGTTGATATTCACTGATGGACTCCAAAGAATCATTA ACTCCTGGTAGAGAAGAAAACCCCAGCAGTGTGCTTGCTCAGGAGAGGGGAGATGTG ATGGACTTCTATAAAACCCTAAGAGGAGGAGCTACTGTGAAGGTTTCTGCGTCTTCA CCCTCACTGGCTGTCGCTTCTCAATCAGACTCCAAGCAGCGAAGACTTTTGGTTGAT TTTCCAAAAGGCTCAGTAAGCAATGCGCAGCAGCCAGATCTGTCCAAAGCAGTTTCA CTCTCAATGGGACTGTATATGGGAGAGACAGAAACAAAAGTGATGGGAAATGACCTG GGATTCCCACAGCAGGGCCAAATCAGCCTTTCCTCGGGGGAAACAGACTTAAAGCTT TTGGAAGAAAGCATTGCAAACCTCAATAGGTCGACCAGTGTTCCAGAGAACCCCAAG AGTTCAGCATCCACTGCTGTGTCTGCTGCCCCCACAGAGAAGGAGTTTCCAAAAACT CACTCTGATGTATCTTCAGAACAGCAACATTTGAAGGGCCAGACTGGCACCAACGGT GGCAATGTGAAATTGTATACCACAGACCAAAGCACCTTTGACATTTTGCAGGATTTG GAGTTTTCTTCTGGGTCCCCAGGTAAAGAGACGAATGAGAGTCCTTGGAGATCAGAC CTGTTGATAGATGAAAACTGTTTGCTTTCTCCTCTGGCGGGAGAAGACGATTCATTC CTTTTGGAAGGAAACTCGAATGAGGACTGCAAGCCTCTCATTTTACCGGACACTAAA CCCAAAATTAAGGATAATGGAGATCTGGTTTTGTCAAGCCCCAGTAATGTAACACTG CCCCAAGTGAAAACAGAAAAAGAAGATTTCATCGAACTCTGCACCCCTGGGGTAATT AAGCAAGAGAAACTGGGCACAGTTTACTGTCAGGCAAGCTTTCCTGGAGCAAATATA ATTGGTAATAAAATGTCTGCCATTTCTGTTCATGGTGTGAGTACCTCTGGAGGACAG ATGTACCACTATGACATGAATACAGCATCCCTTTCTCAACAGCAGGATCAGAAGCCT ATTTTTAATGTCATTCCACCAATTCCCGTTGGTTCCGAAAATTGGAATAGGTGCCAA GGATCTGGAGATGACAACTTGACTTCTCTGGGGACTCTGAACTTCCCTGGTCGAACA GTTTTTTCTAATGGCTATTCAAGCCCCAGCATGAGACCAGATGTAAGCTCTCCTCCA TCCAGCTCCTCAACAGCAACAACAGGACCACCTCCCAAACTCTGCCTGGTGTGCTCT GATGAAGCTTCAGGATGTCATTATGGAGTCTTAACTTGTGGAAGCTGTAAAGTTTTC TTCAAAAGAGCAGTGGAAGGACAGCACAATTACCTATGTGCTGGAAGGAATGATTGC ATCATCGATAAAATTCGAAGAAAAAACTGCCCAGCATGCCGCTATCGAAAATGTCTT C AGGC T GGAAT GAACC T GGAAGC T CGAAAAAC AAAGAAAAAAA AAAAGGAAT T C AG CAGGCCACTACAGGAGTCTCACAAGAAACCTCTGAAAATCCTGGTAACAAAACAATA GTTCCTGCAACGTTACCACAACTCACCCCTACCCTGGTGTCACTGTTGGAGGTTATT GAACCTGAAGTGTTATATGCAGGATATGATAGCTCTGTTCCAGACTCAACTTGGAGG ATCATGACTACGCTCAACATGTTAGGAGGGCGGCAAGTGATTGCAGCAGTGAAATGG GCAAAGGCAATACCAGGTTTCAGGAACTTACACCTGGATGACCAAATGACCCTACTG CAGTACTCCTGGATGTTTCTTATGGCATTTGCTCTGGGGTGGAGATCATATAGACAA TCAAGTGCAAACCTGCTGTGTTTTGCTCCTGATCTGATTATTAATGAGCAGAGAATG ACTCTACCCTGCATGTACGACCAATGTAAACACATGCTGTATGTTTCCTCTGAGTTA CACAGGCTTCAGGTATCTTATGAAGAGTATCTCTGTATGAAAACCTTACTGCTTCTC TCTTCAGTTCCTAAGGACGGTCTGAAGAGCCAAGAGCTATTTGATGAAATTAGAATG ACCTACATCAAAGAGCTAGGAAAAGCCATTGTCAAGAGGGAAGGAAACTCCAGCCAG AACTGGCAGCGGTTTTATCAACTGACAAAACTCTTGGATTCTATGCATGAAGTGGTT GAAAATCTCCTTAACTATTGCTTCCAAACATTTTTGGATAAGACCATGAGTATTGAA TTCCCCGAGATGTTAGCTGAAATCATCACCAATCAGATACCAAAATATTCAAATGGA AATATCAAAAAACTTCTGTTTCATCAAAAGTGACTGCCTTAATAAGAATGGTTGCCT TAAAGAAAGTCGAATTAATAGCTTTTATTGTATAAACTATCAGTTTGTCCTGTAGAG GTTTTGTTGTTTTATTTTTTATTGTTTTCATCTGTTGTTTTGTTTTAAATACGCACT ACATGTGGTTTATAGAGGGCCAAGACTTGGCAACAGAAGCAGTTGAGTCGTCATCAC TTTTCAGTGATGGGAGAGTAGATGGTGAAATTTATTAGTTAATATATCCCAGAAATT AGAAACCTTAATATGTGGACGTAATCTCCACAGTCAAAGAAGGATGGCACCTAAACC ACCAGTGCCCAAAGTCTGTGTGATGAACTTTCTCTTCATACTTTTTTTCACAGTTGG CTGGATGAAATTTTCTAGACTTTCTGTTGGTGTATCCCCCCCCTGTATAGTTAGGAT AGCATTTTTGATTTATGCATGGAAACCTGAAAAAAAGTTTACAAGTGTATATCAGAA AAGGGAAGTTGTGCCTTTTATAGCTATTACTGTCTGGTTTTAACAATTTCCTTTATA TTTAGTGAACTACGCTTGCTCATTTTTTCTTACATAATTTTTTATTCAAGTTATTGT ACAGCTGTTTAAGATGGGCAGCTAGTTCGTAGCTTTCCCAAATAAACTCTAAACATT AATCAATCATCTGTGTGAAAATGGGTTGGTGCTTCTAACCTGATGGCACTTAGCTAT C AG AAG AC C AC AAAAAT T G AC T CAAA CTCCAG ATTCTTGT C AAAAAAAAAAAAAA AAAAGCTCATATTTTGTATATATCTGCTTCAGTGGAGAATTATATAGGTTGTGCAAA TTAACAGTCCTAACTGGTATAGAGCACCTAGTCCAGTGACCTGCTGGGTAAACTGTG GATGATGGTTGCAAAAGACTAATTTAAAAAATAACTACCAAGAGGCCCTGTCTGTAC CTAACGCCCTATTTTTGCAATGGCTATATGGCAAGAAAGCTGGTAAACTATTTGTCT TTCAGGACCTTTTGAAGTAGTTTGTATAACTTCTTAAAAGTTGTGATTCCAGATAAC CAGCTGTAACACAGCTGAGAGACTTTTAATCAGACAAAGTAATTCCTCTCACTAAAC TTTACCCAAAAACTAAATCTCTAATATGGCAAAAATGGCTAGACACCCATTTTCACA TTCCCATCTGTCACCAATTGGTTAATCTTTCCTGATGGTACAGGAAAGCTCAGCTAC TGATTTTTGTGATTTAGAACTGTATGTCAGACATCCATGTTTGTAAAACTACACATC CCTAATGTGTGCCATAGAGTTTAACACAAGTCCTGTGAATTTCTTCACTGTTGAAAA TTATTTTAAACAAAATAGAAGCTGTAGTAGCCCTTTCTGTGTGCACCTTACCAACTT TCTGTAAACTCAAAACTTAACATATTTACTAAGCCACAAGAAATTTGATTTCTATTC AAGGTGGCCAAA A TGTGTAATAGAAAACTGAAAATCTAA A AAAAA ATGG AACTTCTAATATATTTTTATATTTAGTTATAGTTTCAGATATATATCATATTGGTAT TCACTAATCTGGGAAGGGAAGGGCTACTGCAGCTTTACATGCAATTTATTAAAATGA TTGTAAAATAGCTTGTATAGTGTAAAATAAGAATGATTTTTAGATGAGATTGTTTTA TCATGACATGTTATATATTTTTTGTAGGGGTCAAAGAAATGCTGATGGATAACCTAT ATGATTTATAGTTTGTACATGCATTCATACAGGCAGCGATGGTCTCAGAAACCAAAC AGTTTGCTCTAGGGGAAGAGGGAGATGGAGACTGGTCCTGTGTGCAGTGAAGGTTGC TGAGGCTCTGACCCAGTGAGATTACAGAGGAAGTTATCCTCTGCCTCCCATTCTGAC CACCCTTCTCATTCCAACAGTGAGTCTGTCAGCGCAGGTTTAGTTTACTCAATCTCC CCTTGCACTAAAGTATGTAAAGTATGTAAACAGGAGACAGGAAGGTGGTGCTTACAT CCTTAAAGGCACCATCTAATAGCGGGTTACTTTCACATACAGCCCTCCCCCAGCAGT TGAATGACAACAGAAGCTTCAGAAGTTTGGCAATAGTTTGCATAGAGGTACCAGCAA TATGTAAATAGTGCAGAATCTCATAGGTTGCCAATAATACACTAATTCCTTTCTATC C AC AAC AAG AG TTTATT C C AAA AAAAT G AGG AC AT GTTTTTGTTTTCTTT GAAT GCTTTTTGAATGTTATTTGTTATTTTCAGTATTTTGGAGAAATTATTTAATAAAAAA ACAATCATTTGCTTTTTGAATGCTCTCTAAAAGGGAATGTAATATTTTAAGATGGTG TGTAACCCGGCTGGATAAATTTTTGGTGCCTAAGAAAACTGCTTGAATATTCTTATC AATGACAGTGTTAAGTTTCAAAAAGAGCTTCTAAAACGTAGATTATCATTCCTTTAT AGAATGTTATGTGGTTAAAACCAGAAAGCACATCTCACACATTAATCTGATTTTCAT CCCAACAATCTTGGCGCTCAAAAAATAGAACTCAATGAGAAAAAGAAGATTATGTGC ACTTCGTTGTCAATAATAAGTCAACTGATGCTCATCGACAACTATAGGAGGCTTTTC ATTAAATGGGAAAAGAAGCTGTGCCCTTTTAGGATACGTGGGGGAAAAGAAAGTCAT CTTAATTATGTTTAATTGTGGATTTAAGTGCTATATGGTGGTGCTGTTTGAAAGCAG ATTTATTTCCTATGTATGTGTTATCTGGCCATCCCAACCCAAACTGTTGAAGTTTGT AGTAACTTCAGTGAGAGTTGGTTACTCACAACAAATCCTGAAAAGTATTTTTAGTGT TTGTAGGTATTCTGTGGGATACTATACAAGCAGAACTGAGGCACTTAGGACATAACA CTTTTGGGGTATATATATCCAAATGCCTAAAACTATGGGAGGAAACCTTGGCCACCC CAAAAGGAAAACTAACATGATTTGTGTCTATGAAGTGCTGGATAATTAGCATGGGAT GAGCTCTGGGCATGCCATGAAGGAAAGCCACGCTCCCTTCAGAATTCAGAGGCAGGG AGCAATTCCAGTTTCACCTAAGTCTCATAATTTTAGTTCCCTTTTAAAAACCCTGAA AACTACATCACCATGGAATGAAAAATATTGTTATACAATACATTGATCTGTCAAACT TCCAGAACCATGGTAGCCTTCAGTGAGATTTCCATCTTGGCTGGTCACTCCCTGACT GTAGCTGTAGGTGAATGTGTTTTTGTGTGTGTGTGTCTGGTTTTAGTGTCAGAAGGG AAATAAAAGTGTAAGGAGGACACTTTAAACCCTTTGGGTGGAGTTTCGTAATTTCCC AGACTATTTTCAAGCAACCTGGTCCACCCAGGATTAGTGACCAGGTTTTCAGGAAAG GATTTGCTTCTCTCTAGAAAATGTCTGAAAGGATTTTATTTTCTGATGAAAGGCTGT ATGAAAATACCCTCCTCAAATAACTTGCTTAACTACATATAGATTCAAGTGTGTCAA TATTCTATTTTGTATATTAAATGCTATATAATGGGGACAAATCTATATTATACTGTG TATGGCATTATTAAGAAGCTTTTTCATTATTTTTTATCACAGTAATTTTAAAATGTG TAAAAATTAAAACCAGTGACTCCTGTTTAAAAATAAAAGTTGTAGTTTTTTATTCAT GCTGAATAATAATCTGTAGTTAAAAAAAAAGTGTCTTTTTACCTACGCAGTGAAATG TCAGACTGTAAAACCTTGTGTGGAAATGTTTAACTTTTATTTTTTCATTTAAATTTG CTGTTCTGGTATTACCAAACCACACATTTGTACCGAATTGGCAGTAAATGTTAGCCA TTTACAGCAATGCCAAATATGGAGAAACATCATAATAAAAAAATCTGCTTTTTCATT AAAAAAAAAAAAAAAAAA (SEQ ID NO: 2) D. GLUCOCORTICOID RECEPTOR β Real time PCR product (91bp):

AGAACTGGCAGCGGTTTTATCaactgacaaaactcttggattctatgcat

gaaaatgttatgtggttaAAACCAGAAAGCACATCTCACAC (SEQIDNO: 15) Genbank for GR β (transcript variant 6): NM 001020825

GGCGCCGCCTCCACCCGCTCCCCGCTCGGTCCCGCTCGCTCGCCCAGGCCGGGCTGC CCTTTCGCGTGTCCGCGCTCTCTTCCCTCCGCCGCCGCCTCCTCCATTTTGCGAGCT CGTGTCTGTGACGGGAGCCCGAGTCACCGCCTGCCCGTCGGGGACGGATTCTGTGGG TGGAAGGAGACGCCGCAGCCGGAGCGGCCGAAGCAGCTGGGACCGGGACGGGGCACG CGCGCCCGGAACCTCGACCCGCGGAGCCCGGCGCGGGGCGGAGGGCTGGCTTGTCAG CTGGGCAATGGGAGACTTTCTTAAATAGGGGCTCTCCCCCCACCCATGGAGAAAGGG GCGGCTGTTTACTTCCTTTTTTTAGAAAAAAAAAATATATTTCCCTCCTGCTCCTTC TGCGTTCACAAGCTAAGTTGTTTATCTCGGCTGCGGCGGGAACTGCGGACGGTGGCG GGCGAGCGGCTCCTCTGCCAGAGTTGATATTCACTGATGGACTCCAAAGAATCATTA ACTCCTGGTAGAGAAGAAAACCCCAGCAGTGTGCTTGCTCAGGAGAGGGGAGATGTG ATGGACTTCTATAAAACCCTAAGAGGAGGAGCTACTGTGAAGGTTTCTGCGTCTTCA CCCTCACTGGCTGTCGCTTCTCAATCAGACTCCAAGCAGCGAAGACTTTTGGTTGAT TTTCCAAAAGGCTCAGTAAGCAATGCGCAGCAGCCAGATCTGTCCAAAGCAGTTTCA CTCTCAATGGGACTGTATATGGGAGAGACAGAAACAAAAGTGATGGGAAATGACCTG GGATTCCCACAGCAGGGCCAAATCAGCCTTTCCTCGGGGGAAACAGACTTAAAGCTT TTGGAAGAAAGCATTGCAAACCTCAATAGGTCGACCAGTGTTCCAGAGAACCCCAAG AGTTCAGCATCCACTGCTGTGTCTGCTGCCCCCACAGAGAAGGAGTTTCCAAAAACT CACTCTGATGTATCTTCAGAACAGCAACATTTGAAGGGCCAGACTGGCACCAACGGT GGCAATGTGAAATTGTATACCACAGACCAAAGCACCTTTGACATTTTGCAGGATTTG GAGTTTTCTTCTGGGTCCCCAGGTAAAGAGACGAATGAGAGTCCTTGGAGATCAGAC CTGTTGATAGATGAAAACTGTTTGCTTTCTCCTCTGGCGGGAGAAGACGATTCATTC CTTTTGGAAGGAAACTCGAATGAGGACTGCAAGCCTCTCATTTTACCGGACACTAAA CCCAAAATTAAGGATAATGGAGATCTGGTTTTGTCAAGCCCCAGTAATGTAACACTG CCCCAAGTGAAAACAGAAAAAGAAGATTTCATCGAACTCTGCACCCCTGGGGTAATT AAGCAAGAGAAACTGGGCACAGTTTACTGTCAGGCAAGCTTTCCTGGAGCAAATATA ATTGGTAATAAAATGTCTGCCATTTCTGTTCATGGTGTGAGTACCTCTGGAGGACAG ATGTACCACTATGACATGAATACAGCATCCCTTTCTCAACAGCAGGATCAGAAGCCT ATTTTTAATGTCATTCCACCAATTCCCGTTGGTTCCGAAAATTGGAATAGGTGCCAA GGATCTGGAGATGACAACTTGACTTCTCTGGGGACTCTGAACTTCCCTGGTCGAACA GTTTTTTCTAATGGCTATTCAAGCCCCAGCATGAGACCAGATGTAAGCTCTCCTCCA TCCAGCTCCTCAACAGCAACAACAGGACCACCTCCCAAACTCTGCCTGGTGTGCTCT GATGAAGCTTCAGGATGTCATTATGGAGTCTTAACTTGTGGAAGCTGTAAAGTTTTC TTCAAAAGAGCAGTGGAAGGACAGCACAATTACCTATGTGCTGGAAGGAATGATTGC ATCATCGATAAAATTCGAAGAAAAAACTGCCCAGCATGCCGCTATCGAAAATGTCTT C AGGC T GGAAT GAACC T GGAAGC T CGAAAAAC AAAGAAAAAAA AAAAGGAAT T C AG CAGGCCACTACAGGAGTCTCACAAGAAACCTCTGAAAATCCTGGTAACAAAACAATA GTTCCTGCAACGTTACCACAACTCACCCCTACCCTGGTGTCACTGTTGGAGGTTATT GAACCTGAAGTGTTATATGCAGGATATGATAGCTCTGTTCCAGACTCAACTTGGAGG ATCATGACTACGCTCAACATGTTAGGAGGGCGGCAAGTGATTGCAGCAGTGAAATGG GCAAAGGCAATACCAGGTTTCAGGAACTTACACCTGGATGACCAAATGACCCTACTG CAGTACTCCTGGATGTTTCTTATGGCATTTGCTCTGGGGTGGAGATCATATAGACAA TCAAGTGCAAACCTGCTGTGTTTTGCTCCTGATCTGATTATTAATGAGCAGAGAATG ACTCTACCCTGCATGTACGACCAATGTAAACACATGCTGTATGTTTCCTCTGAGTTA CACAGGCTTCAGGTATCTTATGAAGAGTATCTCTGTATGAAAACCTTACTGCTTCTC TCTTCAGTTCCTAAGGACGGTCTGAAGAGCCAAGAGCTATTTGATGAAATTAGAATG ACCTACATCAAAGAGCTAGGAAAAGCCATTGTCAAGAGGGAAGGAAACTCCAGCCAG AACTGGCAGCGGTTTTATCAACTGACAAAACTCTTGGATTCTATGCATGAAAATGTT ATGTGGTTAAAACCAGAAAGCACATCTCACACATTAATCTGATTTTCATCCCAACAA TCTTGGCGCTCAAAAAATAGAACTCAATGAGAAAAAGAAGATTATGTGCACTTCGTT GTCAATAATAAGTCAACTGATGCTCATCGACAACTATAGGAGGCTTTTCATTAAATG GGAAAAGAAGCTGTGCCCTTTTAGGATACGTGGGGGAAAAGAAAGTCATCTTAATTA TGTTTAATTGTGGATTTAAGTGCTATATGGTGGTGCTGTTTGAAAGCAGATTTATTT CCTATGTATGTGTTATCTGGCCATCCCAACCCAAACTGTTGAAGTTTGTAGTAACTT CAGTGAGAGTTGGTTACTCACAACAAATCCTGAAAAGTATTTTTAGTGTTTGTAGGT ATTCTGTGGGATACTATACAAGCAGAACTGAGGCACTTAGGACATAACACTTTTGGG GTATATATATCCAAATGCCTAAAACTATGGGAGGAAACCTTGGCCACCCCAAAAGGA AAACTAACATGATTTGTGTCTATGAAGTGCTGGATAATTAGCATGGGATGAGCTCTG GGCATGCCATGAAGGAAAGCCACGCTCCCTTCAGAATTCAGAGGCAGGGAGCAATTC CAGTTTCACCTAAGTCTCATAATTTTAGTTCCCTTTTAAAAACCCTGAAAACTACAT CACCATGGAATGAAAAATATTGTTATACAATACATTGATCTGTCAAACTTCCAGAAC CATGGTAGCCTTCAGTGAGATTTCCATCTTGGCTGGTCACTCCCTGACTGTAGCTGT AGGTGAATGTGTTTTTGTGTGTGTGTGTCTGGTTTTAGTGTCAGAAGGGAAATAAAA GTGTAAGGAGGACACTTTAAACCCTTTGGGTGGAGTTTCGTAATTTCCCAGACTATT TTCAAGCAACCTGGTCCACCCAGGATTAGTGACCAGGTTTTCAGGAAAGGATTTGCT TCTCTCTAGAAAATGTCTGAAAGGATTTTATTTTCTGATGAAAGGCTGTATGAAAAT ACCCTCCTCAAATAACTTGCTTAACTACATATAGATTCAAGTGTGTCAATATTCTAT TTTGTATATTAAATGCTATATAATGGGGACAAATCTATATTATACTGTGTATGGCAT TATTAAGAAGCTTTTTCATTATTTTTTATCACAGTAATTTTAAAATGTGTAAAAATT AAAACCAGTGACTCCTGTTTAAAAATAAAAGTTGTAGTTTTTTATTCATGCTGAATA ATAATCTGTAGTTAAAAAAAAAGTGTCTTTTTACCTACGCAGTGAAATGTCAGACTG TAAAACCTTGTGTGGAAATGTTTAACTTTTATTTTTTCATTTAAATTTGCTGTTCTG GTATTACCAAACCACACATTTGTACCGAATTGGCAGTAAATGTTAGCCATTTACAGC AATGCCAAATATGGAGAAACATCATAATAAAAAAATCTGCTTTTTCATTA ( SEQ ID NO: 3)

This concludes the description of illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Claims

CLAIMS:

1. A method of examining an individual for a presence or absence of a biomarker observed in irritable bowel syndrome, the method comprising the steps of:

(a) obtaining a biological sample derived from the individual comprising genomic DNA from leukocytes; and

(b) observing cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1;

so that the presence or absence of the biomarker observed in irritable bowel syndrome is examined.

2. The method of claim 1, wherein cytosine methylation is observed in at least one cytosine nucleotide selected from the group consisting of CpG site 1, 2, 3, 4, 5, 6, 7 or 8 in SEQ ID NO: 1.

3. The method of claim 2, wherein cytosine methylation is observed in 2, 3, 4, 5, 6, 7 or 8 cytosine nucleotides selected from the group consisting of CpG site 1, 2, 3, 4, 5, 6, 7 or 8 in SEQ ID NO: 1.

4. The method of claim 3, wherein:

at least 20 % cytosine methylation at CpG site 1, 2, 3, 7 or 8 provides evidence of irritable bowel syndrome;

at least 50 % cytosine methylation at CpG site 1, 2 or 3 provides evidence of irritable bowel syndrome with diarrhea subtype; and/or

at least 50 % cytosine methylation at CpG site 7 or 8 provides evidence of irritable bowel syndrome with constipation subtype.

5. The method of claim 4, wherein: less than 20 % cytosine methylation at CpG site 7 or 8 provides evidence of irritable bowel syndrome with diarrhea subtype; and/or

less than 50 % cytosine methylation at CpG site 1, 2 or 3 provides evidence of irritable bowel syndrome with constipation subtype.

6. The method of claim 1, wherein:

the biological sample comprises mRNA; and

the method further comprises characterizing expression levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA in the leukocytes.

7. The method of claim 1, wherein a bisulfite conversion process is performed so that cytosine residues in the genomic DNA are transformed to uracil, while 5- methylcytosine residues in the genomic DNA are not transformed to uracil.

8. The method of claim 1, wherein the genomic DNA is amplified by a polymerase chain reaction process.

9. The method of claim 1, further comprising administering a therapeutic agent useful to treat a symptom of irritable bowel syndrome to an individual identified as expressing a biomarker observed in irritable bowel syndrome.

10. A method of obtaining information for determining a prognosis or therapy for a patient having or suspected of having irritable bowel syndrome, the method comprising:

(a) observing a level of glucocorticoid receptor mRNA expressed in leukocytes obtained from the patient;

(b) comparing the level of glucocorticoid receptor mRNA expressed in leukocytes obtained from the patient with a level of glucocorticoid receptor a mRNA expressed in control leukocytes obtained from an individual not having irritable bowel syndrome; and

(c) correlating the level of glucocorticoid receptor observed in the leukocytes obtained from the patient with a prognosis or therapy for irritable bowel syndrome.

11. The method of claim 10, wherein the expression of glucocorticoid receptor a mRNA (SEQ ID NO: 2) and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA is examined.

12. The method of claim 11, further comprising correlating the level of glucocorticoid receptor β mRNA observed in the leukocytes obtained from the patient with a irritable bowel syndrome subtype selected from the group consisting of irritable bowel syndrome with constipation and irritable bowel syndrome with diarrhea.

13. The method of claim 11 , wherein:

expression levels of glucocorticoid receptor a mRNA (SEQ ID NO: 2) in the patient leukocytes that are at least X% below the mean levels of glucocorticoid receptor a mRNA (SEQ ID NO: 2) expressed in the control leukocytes provides evidence of irritable bowel syndrome.

14. The method of claim 11 , wherein:

expression levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) in the patient leukocytes that are at least X% above the mean levels of glucocorticoid receptor β mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with constipation.

15. The method of claim 11, wherein the level of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor β (SEQ ID NO: 3) mRNA is observed using a real time polymerase chain reaction process.

16. The method of claim 10, further comprising observing a pattern of cytosine methylation in a genomic glucocorticoid receptor DNA sequence comprising SEQ ID NO: 1.

17. A kit for examining a biomarker observed in irritable bowel syndrome, wherein the kit comprises:

a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor NR3C1 promoter (SEQ ID NO: 1); or

a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor a mRNA (SEQ ID NO: 2) or glucocorticoid receptor β (SEQ ID NO: 3) mRNA; or

a polynucleotide probe that hybridizes to glucocorticoid receptor a mRNA (SEQ ID NO: 2) or glucocorticoid receptor β (SEQ ID NO: 3) mRNA; and

a reagent selected for use in:

a genomic DNA polymerization process;

a genomic DNA hybridization process;

a genomic DNA bisulfite conversion process; or

a reverse transcription process.

18. The kit of claim 17, wherein the kit comprises a plurality of primers that amplify a polynucleotide sequence of glucocorticoid receptor NR3C1 promoter (SEQ ID NO: 1) and a reagent used in a genomic DNA bisulfite conversion process.

19. The kit of claim 17, wherein the kit comprises a plurality of primer sets for amplifying glucocorticoid receptor a mRNA (SEQ ID NO: 2) and glucocorticoid receptor β (SEQ ID NO: 3) and a reagent used in a reverse transcription process.

20. The kit of claim 17, wherein the kit comprises a polynucleotide probe that hybridizes to glucocorticoid receptor a mRNA (SEQ ID NO: 2) and a polynucleotide probe that hybridizes to glucocorticoid receptor β (SEQ ID NO: 3) mRNA.