WO2013082288A1 - Méthylation du gène récepteur des glucocorticoïdes nr3c1 dans un syndrome de l'intestin irritable et autres troubles de stress - Google Patents

Méthylation du gène récepteur des glucocorticoïdes nr3c1 dans un syndrome de l'intestin irritable et autres troubles de stress Download PDF

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WO2013082288A1
WO2013082288A1 PCT/US2012/067069 US2012067069W WO2013082288A1 WO 2013082288 A1 WO2013082288 A1 WO 2013082288A1 US 2012067069 W US2012067069 W US 2012067069W WO 2013082288 A1 WO2013082288 A1 WO 2013082288A1
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seq
mrna
glucocorticoid receptor
bowel syndrome
irritable bowel
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Lin Chang
Emeran A. MAYER
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to methods of detecting and analyzing patterns of cytosine methylation and mRNA expression that are associated with pathological syndromes.
  • 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).
  • IBS Irritable bowel syndrome
  • irritable bowel syndrome is currently a symptom-based disorder.
  • GR NR3C1 glucococorticoid receptor
  • 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.
  • NR3C1 promoter methylation was examined in peripheral blood T lymphocytes obtained from either Rome III+ IBS patients or age- and sex-matched healthy controls.
  • 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.
  • certain GR isoform mRNA profiles were observed to correlate with IBS.
  • 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.
  • 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.
  • 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.
  • the method is performed on an individual previously selected as having one or more signs or symptoms characteristic of irritable bowel syndrome.
  • 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 1 7 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.
  • 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.
  • an observation of an at least 20 % cytosine methylation at CpG site 1, 2, 3, 7 or 8 provides evidence of irritable bowel syndrome.
  • 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.
  • 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.
  • a plurality of IBS biomarkers disclosed herein can be examined in methods of the invention.
  • the method in addition to observing methylation patterns in the NR3C1 exon 1 7 promoter region, the method further comprises characterizing expression levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor ⁇ (SEQ ID NO: 3) mRNA in the leukocytes.
  • inventions 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 ⁇ (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.
  • glucocorticoid receptor a mRNA SEQ ID NO: 2
  • 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.
  • expression levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) in the patient leukocytes that are at least X% above the mean levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with constipation.
  • the methods of the invention can be adapted for use with a variety of art accepted processes.
  • 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.
  • the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process.
  • the genomic DNA is hybridized to a complimentary sequence (e.g. a synthetic polynucleotide sequence) that is coupled to a matrix (e.g.
  • glucocorticoid receptor a SEQ ID NO: 2
  • glucocorticoid receptor ⁇ SEQ ID NO: 3
  • kits for obtaining information useful in diagnosing IBS and/or a IBS bowel habit subtype include 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.
  • 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.
  • 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.
  • kits 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 ⁇ (SEQ ID NO: 3) mRNA and a reagent selected for use in a RT-PCR process (e.g. reverse transcriptase).
  • kits 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 ⁇ (SEQ ID NO: 3) mRNA.
  • 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.
  • Figure 4 provides a schematic showing the DNA sequence of the NR3C1 promoter area that includes 8 CpG sites examined for their methylation status.
  • genomic 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.
  • epigenetic 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.
  • nucleic acids 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.
  • 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.
  • 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.
  • probes are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid.
  • probe 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.
  • label 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.
  • primer 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.
  • complementary 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%.
  • complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • 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.
  • hybridization 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.
  • array 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.
  • biomarker 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.
  • leukocyte 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.
  • 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.
  • 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.
  • 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).
  • GI organic gastrointestinal
  • 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.
  • the associated molecular signatures include increased methylation of the NR3C1 exon 1 7 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).
  • 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).
  • GRa active
  • GPP inhibitory
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • bowel habit predominance was associated with distinct differences in the methylation pattern of the NR3C1 promoter and expression of GR.
  • decreased GR expression was associated with a greater Cortisol response to a stressful visceral stimulus in patients, but not healthy controls.
  • 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.
  • 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.
  • the leukocytes are obtained from the peripheral blood of the patient.
  • the leukocytes are obtained from the saliva of the patient.
  • 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 1 7 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.
  • 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.
  • 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).
  • an at least 50, 60, 70 or 80 % cytosine methylation at CpG site 1, 2 or 3 provides evidence of a irritable bowel syndrome diarrhea subtype (see, e.g. Figure 1).
  • an at least 50, 60 or 70 % cytosine methylation at CpG site 7 or 8 provides evidence of a irritable bowel syndrome constipation subtype (see, e.g. Figure 1).
  • 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).
  • the method further comprises characterizing expression levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor ⁇ (SEQ ID NO: 3) mRNA in leukocytes.
  • DNA samples e.g. 1 ⁇ g genomic DAN obtained from a peripheral blood monocyte
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IBD inflammatory bowel disease
  • celiac disease celiac disease
  • the expression profile of glucocorticoid receptor a mRNA (SEQ ID NO: 2) and/or glucocorticoid receptor ⁇ (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.
  • 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.
  • expression levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) in the patient leukocytes that are at least 10% above the mean levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with constipation.
  • expression levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) in the patient leukocytes that are below at least 10% below the mean levels of glucocorticoid receptor ⁇ mRNA (SEQ ID NO: 3) expressed in the control leukocytes provides evidence of irritable bowel syndrome with diarrhea.
  • glucocorticoid receptor ⁇ mRNA SEQ ID NO: 3
  • Expression levels of glucocorticoid receptor ⁇ 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 ⁇ 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.
  • 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).
  • tricyclic antidepressants e.g., Elavil
  • selective serotonin reuptake inhibitors e.g. Celexa
  • serotonin- norepinephrine reuptake inhibitors e.g., Cymbalta
  • 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.
  • the therapeutic agent is selected following the observation of an IBS biomarker profile that is characteristic of a specific bowel habit subtype.
  • a patient identified as having Irritable Bowel Syndrome with a constipation subtype is administered Lubiprostone.
  • the IBS biomarker profile can predict treatment response.
  • a therapeutic regimen is selected following the observation of the IBS biomarker profile that is associated with a symptomatic response to this treatment.
  • the methods of the invention can be adapted for use with a variety of art accepted processes.
  • 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.
  • the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process.
  • 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).
  • the levels of glucocorticoid receptor a (SEQ ID NO: 2) mRNA and/or glucocorticoid receptor ⁇ (SEQ ID NO: 3) mRNA are observed using a real time polymerase chain reaction process.
  • kits for obtaining information useful to determine the age of an individual include 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.
  • 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).
  • 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).
  • a genomic DNA polymerization process e.g. a composition comprising a mixture of dNTPs
  • a genomic DNA hybridization process e.g. a genomic DNA hybridization process
  • a genomic DNA bisulfite conversion process e.g. a sodium metabisulfate composition
  • kits 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 ⁇ (SEQ ID NO: 3) mRNA and a reagent selected for use in a RT-PCR process (e.g. a reverse transcriptase).
  • a reagent selected for use in a RT-PCR process e.g. a reverse transcriptase
  • kits 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 ⁇ (SEQ ID NO: 3) mRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SBE single base extension
  • MIP Molecular Inversion Probe
  • 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).
  • a complimentary sequence e.g. a synthetic polynucleotide sequence
  • a matrix e.g. one disposed within a microarray
  • the genomic DNA is transformed from its natural state via amplification by a polymerase chain reaction process.
  • 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.
  • EXAMPLE 1 EXPERIMENTAL PROTOCOLS SHOWING TYPICAL
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 ⁇ g) 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 ⁇ g) 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 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.
  • DNA methylation status of eight CpG sites in the NR3C1 promoter are shown in Figure 4.
  • NR3C1 methylation was not present at any of the CpG sites.
  • 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).
  • 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 ⁇ -Actin and GAPDH.
  • 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.
  • 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.
  • IBS-C- IBS with constipation IBS-D- IBS with diarrhea
  • 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
  • Genbank Accession Numbers for GRa are (transcript variants 1,2,3,4,5,7): NM_001204264 (for variant 1 )

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Abstract

La présente invention identifie des sites dans le promoteur de récepteur des glucocorticoïdes (GR) pour lesquels le degré de méthylation de cytosine dans les leucocytes peut être corrélé avec un syndrome de l'intestin irritable (IBS) et un sous-type de l'IBS. La présente invention identifie également des profils d'ARNm GR-a et GR-β dans des leucocytes de sang périphérique qui peuvent être corrélés avec l'IBS et un sous-type de l'IBS. L'invention concerne des procédés et des matières qui utilisent l'observation de la méthylation de l'ADN et/ou l'expression de l'ARNm pour obtenir des informations se rapportant au syndrome de l'intestin irritable (IBS).
PCT/US2012/067069 2011-11-29 2012-11-29 Méthylation du gène récepteur des glucocorticoïdes nr3c1 dans un syndrome de l'intestin irritable et autres troubles de stress WO2013082288A1 (fr)

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WO2019232468A1 (fr) * 2018-05-31 2019-12-05 The Regents Of The University Of California Biomarqueurs basés sur la méthylation de l'adn pour le syndrome du côlon irritable et la maladie du côlon irritable
CN111051534A (zh) * 2017-08-30 2020-04-21 百时美施贵宝公司 监测体内施用糖皮质激素介导的药效学反应的方法

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CN111051534A (zh) * 2017-08-30 2020-04-21 百时美施贵宝公司 监测体内施用糖皮质激素介导的药效学反应的方法
WO2019232468A1 (fr) * 2018-05-31 2019-12-05 The Regents Of The University Of California Biomarqueurs basés sur la méthylation de l'adn pour le syndrome du côlon irritable et la maladie du côlon irritable

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