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WO2009060089A1 - Procédé de diagnostic et d'analyse de sous-groupe dans des sujets ayant ou étant suspectés d'avoir le syndrome du côlon irritable, acides nucléiques et kits, et leur utilisation - Google Patents

Procédé de diagnostic et d'analyse de sous-groupe dans des sujets ayant ou étant suspectés d'avoir le syndrome du côlon irritable, acides nucléiques et kits, et leur utilisation Download PDF

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Publication number
WO2009060089A1
WO2009060089A1 PCT/EP2008/065182 EP2008065182W WO2009060089A1 WO 2009060089 A1 WO2009060089 A1 WO 2009060089A1 EP 2008065182 W EP2008065182 W EP 2008065182W WO 2009060089 A1 WO2009060089 A1 WO 2009060089A1
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Prior art keywords
receptor
subject
nucleotide sequence
irritable bowel
receptor gene
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Inventor
Beate Niesler
Gudrun Rappold
Johannes Kapeller
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Universitaetsklinikum Heidelberg
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Universitaetsklinikum Heidelberg
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/156—Polymorphic or mutational 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the invention relates to a method of diagnosis and subgroup analysis in subjects having or being suspected of having Irritable Bowel Syndrome based on the detection of polymorphisms in serotonin type 3 receptor (5-HT3) genes.
  • the invention relates to certain nucleic acids, in particular probes, and kits containing said nucleic acids.
  • the methods, probes and kits are useful for diagnosing Irritable Bowel Syndrome and predicting the potential benefits of 5-HT3 receptor antagonist treatment.
  • IBS Irritable Bowel Syndrome
  • IBS-C constipation
  • IBS-D diarrhea
  • IBS- M diarrhea
  • IBS-U unsubtyped Irritable Bowel Syndrome
  • IBS Irritable Bowel Syndrome
  • the serotonin 3 (5-HT3) receptor is a Cys-loop ligand gated ion channel (LGIC) composed of five subunits. It is an important mediator of the action of 5-HT, and has been shown to play a key role in the motor-sensory function and secretion of the gut (Ger- shon, M. D., and Tack, J. 2007.
  • the serotonin signaling system from basic understanding to drug development for functional Gl disorders. Gastroenterology 132:397-414).
  • 5-HT3 receptors are located on peripheral nerve terminals of both vagal and spinal primary afferent neurons innervating the gut, as well as on myenteric and submucosal neurons. They have also been described in the spinal cord and throughout the brain, mostly in the limbic and cortical regions.
  • HTR3A, HTR3B, HTR3C, HTR3D and H7R3£ (WO2006021343; WO2006021347; Niesler, B., Frank, B., Kapeller, J., and Rappold, G.A. 2003. Cloning, physical mapping and ex- pression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E. Gene 310:101 -1 11 ).
  • the 5-HT3A subunit seems to have a key function in the formation of the 5-HT3 receptor since it is the only subunit that can generate functional homopentamers.
  • a -42C>T variant (the C.-42T allele) was shown to be located in the 5' untranslated region of the 5-HT3A receptor gene.
  • Luciferase reporter gene analysis of -42C>T revealed an increased expression compared to the wild-type control.
  • the variant was found to be also associated with bipolar affective disorder, harm avoidance in women and modulation of amygdaloid activity (Niesler, B., Flohr, T., Nothen, M. M., Fischer, C, Rietschel, M., Franzek, E., Albus, M., Propping, P., and Rappold, G.A. 2001. Association between the 5' UTR variant C178T of the serotonin receptor gene HTR3A and bipolar affective disorder. Pharmacogenetics 1 1 :471-475).
  • HTR3A 5 ' UTR variant -42OT and the HTR3E 3 ' UTR variant * 76G>A were found associated with the IBS- D subtype. Functional studies showed that both variants lead to significant upregulation of subunit expression. In HEK293 cells, the HTR3A variant -42OT results in a higher density of 5-HT3A receptors at the cell surface compared to the wild-type control.
  • the HTR3E variant * 76G>A affects the microRNA (miRNA) binding site hsa-miR-510 and leads to a higher luciferase reporter gene expression. Both HTR3E and the miRNA co- localize in enterocytes of the mucosal cell layer of the gut epithelium as shown by in situ hybridization. Moreover, the HTR3C CDS (CoDing Sequence) variant 489A>C (the C.489C allele) was found associated with the IBS-D subtype.
  • the invention provides an improved diagnosis and therapy of Irritable Bowel Syndrome.
  • the SNPs are useful as biomarkers.
  • Specific 5-HT3 receptor antagonists e.g., interfering RNAs or antibodies
  • the present invention relates to a method of diagnosing Irritable Bowel Syndrome in a subject, which comprises
  • * 76) is an adenine; and (iii) a CDS region of a 5-HT3C receptor gene wherein the base at position 489 (c.489) is a cytosine, the presence of said base(s) indicating that the subject has Irritable Bowel Syndrome or may be at risk of developing Irritable Bowel Syndrome.
  • the present invention also relates to a method of determining whether a subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a 5- HT3 receptor antagonist, which comprises (I) obtaining a sample of DNA from the subject; and (II) determining whether said DNA comprises one or more than one sequence selected from the group consisting of (i) a 5' untranslated region of a 5-HT3A receptor gene wherein the base at position -42 (c-42) is a thymine; (ii) a 3' untranslated region of a 5-HT3E receptor gene wherein the base at position * 76 (c.
  • * 76) is an adenine and/or (iii) a CDS region of a 5-HT3C receptor gene wherein the base at position 489 (c.489) is a cytosine, the presence of said base(s) indicating the subject's potential benefit.
  • the methods of the present invention thus comprise obtaining a sample of the subject's DNA and determining the genotype of the subject at polymorphic allelic site(s) in the 5- HT3A, 5-HT3C and/or 5-HT3E receptor gene(s).
  • a first polymorphic allelic site of the present invention is characterized by a cytosine to thymine change (when compared to the wildtype sequence, i.e.
  • HTR3A 5'UTR variant -42OT also referred to as HTR3A C.-42T
  • HTR3A C.-42T a second polymorphic allelic site of the present invention is characterized by a guanine to adenine change (when compared to the wildtype sequence, i.e. the sequence of the more frequent allele) at position * 76 in the 3' untranslated region of the 5-HT3E receptor gene (the so-called HTR3E 3'UTR variant * 76G>A; also referred to as HTR3E c. * 76A).
  • a third polymorphic allelic site of the present invention is characterized by an adenine to cytosine change (when compared to the reference sequence comprising the adenine) at position 489 in the CDS region of the 5-HT3C receptor gene (hereinafter referred to as the HTR3C CDS variant 489A>C or HTR3C C.489C).
  • SNPs single nucleotide polymorphisms
  • methods of the present invention comprise determining the genotype of the subject at two polymorphic allelic sites, i.e., in the 5-HT3A receptor gene and the 5- HT3E receptor gene, in the 5-HT3A receptor gene and the 5-HT3C receptor gene, or in the 5-HT3E receptor gene and the 5-HT3C receptor gene.
  • methods of the present invention comprise determining the genotype of the subject at all three polymorphic allelic sites.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polynucleotide or polypeptide sequences, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1 ): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md.
  • 20894 Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following:(1 ) Algorithm: Needleman and Wunsch, J. MoI Biol.
  • genotyping a subject (or DNA sample) for a polymorphic allele at a defined genomic locus or "determining the genotype at a polymorphic allelic site” means detecting which forms of the allele are present in a subject (or a sample).
  • an individual may be heterozygous or homozygous for a particular allele. More than two forms of an allele may exist, as is the case with microsatellite markers; thus there may be more than three possible genotypes.
  • a "genetic subset" of a population consists of those members of the population having a particular genotype.
  • a population can potentially be divided into three subsets: homozygous for allele 1 , heterozygous, and homozygous for allele 2.
  • HTR3A 5'UTR variant -42OT i.e., the C.-42T allele
  • HTR3E 3'UTR variant * 76G>A i.e., the c.
  • HTR3C CDS variant 489A>C i.e., the C.489C allele
  • two polymorphisms i.e., of the HTR3A 5'UTR variant -42OT and the HTR3E 3'UTR variant * 76G>A, of the HTR3A 5'UTR variant -42OT and the HTR3C CDS variant 489A>C, or of the HTR3E 3'UTR variant * 76G>A and the HTR3C CDS variant 489A>C, or the presence of all three variants, i.e., of the HTR3A 5'UTR variant - 42OT, the HTR3E 3'UTR variant * 76G>A and the HTR3C CDS variant 489A>C, indicates that the subject is likely having Irritable Bowel Syndrome and/or the subject's potential benefit, and administration of 5-HT3 receptor antagonists can be restricted to the polymorph
  • the likelihood of having Irritable Bowel Syndrome and/or the potential benefit from treatment with a 5-HT3 receptor antagonist is higher than in subjects which are heterozygous for said variants. This is especially true for the HTR3C variant.
  • a "benefit” is any amelioration in relevant clinical parameters or decrease in subjective suffering of the subject amenable to scoring, as well as any retardation in the progress of the disease in comparison to an untreated control, that can be causally connected to a therapeutic measure.
  • a “potential benefit” is thus any such benefit that may be expected to be achieved, with a reasonable chance of success, by the intended treatment under the conditions as determined, for an individual subject.
  • a benefit or potential benefit may refer to any extension or reasonably expectable extension, respectively, of life expectancy and/or increase or reasonably expectable increase in the quality- adjusted life years or disability-adjusted life years (QALYs and DALYs).
  • a further aspect of the present invention is a method of treating a subject with Irritable Bowel Syndrome by administering a 5-HT3 receptor antagonist, where the pa- tients have one, two or all of said polymorphisms (variants) in the 5-HT3A, 5-HT3C and/or 5-HT3E gene that is predictive of a higher incidence of relief of IBS symptoms or a lower incidence of side effects when treated with a 5-HT3 receptor antagonist.
  • the incidence of relief is increased (and of side effects decreased) compared to subjects who do not have said polymorphism(s) at the same site of said 5-HT3 genes.
  • 5-HT3 receptors are involved in a wider range of cellular processes than only in the motor-sensory function and secretion of the gut. For instance, the regulation of cognition and emotion has been reported to be influenced by 5-HT3 receptors, said regulation in fact comprising a complex network of interactions and feedbacks.
  • many existing 5-HT3 receptor antagonists, such as alosetron are burdened with a propensity for severe side effects.
  • pharmacological inhibition of 5-HT3 receptors will result in generally pleiotropic effects, many of which may be unwanted or even dangerous, even if these inhibitors are pharmacologically restricted, by use of a drug targeting system or otherwise, to the tissue to be treated.
  • one advantage of the present invention is to be able to restrict 5-HT3 receptor antagonist treatment to cases of verifiable need.
  • a "side effect" is an undesirable response to the administration of a therapeutic compound, i. e., an effect that is not directed to alleviating the symptoms or cause of the disease being treated. Side effects range from minor inconveniences to more serious events.
  • a “5-HT3 receptor antagonist” is a substance which is capable of significantly reducing, under the conditions of interest, the functional activity of any 5-HT3 receptor.
  • 5-HT3 receptor is meant to describe a neurotransmitter-gated ion channel that is believed to consist of an arrangement of five subunits surrounding a central ion- conducting pore. Said subunits include, but are not limited to, the 5-HT3A, 5-HT3B, 5- HT3C, 5-HT3D and 5-HT3E receptor subunits.
  • a 5-HT3 receptor comprises at least one 5-HT3A receptor subunit.
  • a 5-HT3 receptor comprises at least one 5- HT3E receptor subunit.
  • a 5-HT3 receptor comprises at least one 5-HT3C receptor subunit.
  • a 5-HT3 receptor comprises at least one 5-HT3A receptor subunit and at least one 5-HT3E receptor subunit. According to a further embodiment of the invention, a 5-HT3 receptor comprises at least one 5-HT3A receptor subunit and at least one 5-HT3C receptor subunit. According to a further embodiment of the invention, a 5-HT3 receptor comprises at least one 5-HT3C receptor subunit and at least one 5-HT3E receptor subunit. According to a further embodiment of the invention, a 5-HT3 receptor comprises at least one 5-HT3A receptor subunit, at least one 5-HT3C receptor subunit and at least one 5-HT3E receptor subunit.
  • a "5-HT3A receptor subunit” is a protein comprising an amino acid sequence with an overall sequence identity of at least 70 %, preferably at least 80 %, more preferably at least 90 % or at least 95 % and most preferably at least 99 % with the human 5-HT3A receptor subunit, e.g., the protein having the sequence as laid down in SEQ ID NO:8 (NM_000869) or as depicted in Figure 6.
  • a "5-HT3E receptor subunit” is a protein comprising an amino acid sequence with an overall sequence identity of at least 70 %, preferably at least 80 %, more preferably at least 90 % or at least 95 % and most preferably at least 99 % with the human 5-HT3E receptor subunit, e.g., the protein having the sequence as laid down in SEQ ID NO:9 (NM_182589) or as depicted in Figure 7.
  • a "5-HT3C receptor subunit” is a protein comprising an amino acid sequence with an overall sequence identity of at least 70 %, preferably at least 80 %, more preferably at least 90 % or at least 95 % and most preferably at least 99 % with the human 5-HT3C receptor subunit, e.g., the protein having the sequence as laid down in SEQ ID NO:11 (NM_130770) or as depicted in Figure 8.
  • the 5-HT3 receptor antagonist is selected from the group consisting of pro- teins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which is capable of decreasing the functional activity of a 5-HT3 receptor, for instance by decreasing the expression of the 5-HT3 receptor, by post- translationally modifying the 5-HT3 receptor, or by directly interacting with the 5-HT3 receptor.
  • “Functional activity" of 5-HT3 receptors refers to their ability to conduct and control ion movements across cellular membranes resulting in measurable changes in (1 ) the current, (2) the membrane potential, (3) the change in concentration of the transported ions and (4) any other measurable change exerted by 5-HT3 receptors.
  • WO 95/032209 (azabicycloalkyl derivatives of imidazo[1 ,5-a]indol-3-one;
  • WO 95/032204 imidazolylalkyl derivatives of imidazo(1 ,5-a)indol-3-ones
  • WO 95/01 1245 indole derivatives
  • WO 94/012494 dimethylbenzofurans and dimethylbenzopyrans
  • WO 94/01 1347 phenyl imidazolidinone derivatives
  • WO 93/025555 (imidazo[5,1-c][1 , 4]benzoxacin-1-ones)
  • WO 93/018025 (N,N'-disubstituted amide derivatives);
  • WO 93/007147 (3,9-diazabicyclo(3.3.1 )nonane derivatives);
  • WO 92/015593 imidazopyridines;
  • WO 92/015590 meso-azacyclic aromatic acid amides and esters;
  • WO 91/017161 isochinoline amides and esters
  • WO 91/007402 azabicyclo amides and esters
  • WO 90/006309 heteroazabenzobicyclic carboxamides
  • EP-A 621 271 (benzoxazole derivatives);
  • EP-A 573 360 (pyrrolothienopyrazines);
  • EP-A 558 923 (diazabicyclo derivatives);
  • EP-A 560 604 (cinnoline-3-carboxylic acid derivatives; EP-A 422 846 (aroyl-ureas);
  • EP-A 392 663 (carboline derivatives);
  • EP-A 345 956 (tricyclic ketones);
  • EP-A 315 390(4-oxobenzotriazines and 4-oxoquinazolines);
  • EP-A 306 323 tricyclic lactams
  • EP-A 297 651 anellated indole derivatives
  • the 5-HT3 receptor antagonist is selected from the group consisting of alosetron, azasetron, bemesetron, BRL-46470, cilansetron, clozapine, dolasetron, fabesetron, galdansetron, GR-65630, granisetron, ICS-205-930, indisetron, itasetron, lerisetron, lurosetron, LY-278,584, MDL-72222, ondansetron, pa- lonosetron, quipazine, ramosetron, renzapride, ricasetron, SDZ 206-830, tropisetron, Y-25130, zacopride, zatosetron, and pharmaceutically acceptable salts thereof.
  • the 5-HT3 receptor antagonist is an antibody that binds to the 5-HT3 receptor.
  • the 5-HT3 receptor antagonist is an an- tisense oligonucleotide, preferably an antisense RNA specific for a 5-HT3A, 5-HT3C and/or 5-HT3E receptor gene.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nu- cleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as de- scribed above to decrease the level of 5-HT3A, 5-HT3C and/or 5-HT3E gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated syn- thesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioat.es, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of 5-HT3A, 5-HT3C or 5-HT3E gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the 5-HT3A, 5-HT3C or 5-HT3E gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base- pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a 5-HT3A, 5-HT3C or 5-HT3E polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent 5-HT3A, 5-HT3C or 5-HT3E nucleotides, can provide sufficient targeting specificity for 5-HT3A, 5-HT3C or 5-HT3E mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1 , 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular 5-HT3A, 5-HT3C or 5-HT3E polynucleotide sequence.
  • Antisense oligonu- cleotides can be modified without affecting their ability to hybridize to a 5-HT3A, 5- HT3C or 5-HT3E polynucleotide.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art.
  • the 5-HT3 receptor antagonist is a ri- bozyme.
  • Ribozymes are RNA molecules with catalytic activity. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a 5-HT3A, 5-HT3C or 5-HT3E polynucleotide can be used to generate ri- bozymes which will specifically bind to mRNA transcribed from a 5-HT3A, 5-HT3C or 5- HT3E polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a 5-HT3A, 5-HT3C or 5-HT3E RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate 5-HT3A, 5-HT3C or 5-HT3E RNA targets also can be evaluated by testing accessibility to hybridization with com- plementary oligonucleotides using ribonuclease protection assays.
  • nucleotide sequences shown in SEQ ID NO:1 , SEQ ID NO:10 or SEQ ID NO:2 and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct.
  • Mechanical meth- ods such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease 5-HT3A, 5-HT3C or 5-HT3E expression.
  • the construct can be supplied on a plasmid and maintained as a separate element or inte- grated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641 ,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • the 5-HT3 receptor antagonist is a nucleic acid molecule capable of mediating RNA interference (RNAi) against 5-HT3A, 5- HT3C and/or 5-HT3E receptor gene expression, such as a short interfering RNA (siRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA).
  • RNAi RNA interference
  • siRNA short interfering RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • MiRNAs are small regulatory RNAs that control gene expression.
  • the miRNA is complementary or partially complementary to a portion of a target gene or nucleotide sequence and functions to modulate expression of the target sequence or gene.
  • a molecule e.g. a miRNA, comprising the nucleotide sequence: 3 ' acacuaacGGUGAGAGGAUUCAU 5 ' (SEQ ID NO:5) may be used.
  • the sequence corresponds to the sequence of hsa-miR-510 (miR-510; MI0003197) with the exception of a cytosine to uracil change so that SEQ ID NO:5 is complementary to the binding site for miR-510 in the HTR3E * 76G>A variant.
  • a molecule e.g. a miRNA, comprising the nucleotide sequence: 5 ' uacuuaggagaguggcaaucac 3 ' (SEQ ID NO:6) may be used.
  • the sequence has been adapted from database entry MIMAT0002882 (mature sequence).
  • a molecule, e.g. a miRNA, comprising the nucleotide sequence: 5 ' GUGGUGUCCUACUUAGGAGAGUGGCAAUCACAUGUAAU UAGGUGUGAUUGAA ACCUCUAAGAGUGGAGUAACAC 3 ' (SEQ ID NO:7) may be used.
  • the sequence has been adapted from database entry MI0003197 (stem- loop sequence).
  • any pharmacologically acceptable 5-HT3 receptor antagonist may be used in the treatment of Irritable Bowel Syndrome.
  • 5-HT3 receptor antagonists which are capable of significantly reducing, under the conditions of interest, the functional activity of a 5-HT3 receptor that comprises at least one 5-HT3A receptor subunit, a 5-HT3 receptor that comprises at least one 5-HT3E receptor subunit, a 5- HT3 receptor that comprises at least one 5-HT3C receptor subunit, a 5-HT3 receptor that comprises at least one 5-HT3A receptor subunit and at least one 5-HT3E receptor subunit, a 5-HT3 receptor that comprises at least one 5-HT3A receptor subunit and at least one 5-HT3C receptor subunit, a 5-HT3 receptor that comprises at least one 5- HT3C receptor subunit and at least one 5-HT3E receptor subunit, or a 5-HT3 receptor that comprises at least one 5-HT3A receptor subunit, at least one 5-HT3C receptor subunit and at least one 5-HT3E receptor subunit.
  • the 5-HT3 receptor antagonist is 5-HT3 receptor subunit-specific antagonist.
  • Such antagonists include, but are not limited to, antibodies that specifically bind to the 5-HT3A, 5-HT3C and/or 5-HT3E receptor sub- unit.
  • the miRNAs disclosed above are examples of 5-HT3E receptor subunit- specific antagonists.
  • Irritable Bowel Syndrome is a functional bowel disorder characterized by one or more than one of the following symptoms: abdominal pain, discomfort, disor- dered defecation, change in bowel habit. Discomfort means an uncomfortable sensation not described as pain. It is the most common diagnosis made by gastroenterolo- gists. Formal diagnosis is based upon a constellation of symptoms defined by either the Manning or Rome Criteria, in particular the Rome III Criteria (Longstreth G. F. et al., Gasteroenterology 2006, 130,: 1480-1491 ).
  • the Irritable Bowel Syndrome is Irritable Bowel Syndrome without constipation, in particular Irritable Bowel Syndrome with diarrhea (IBS-D), and more particularly diarrhea-predominant Irritable Bowel Syndrome.
  • the subject is a female subject. This embodiment especially refers to the methods, kits and uses of the present invention which are based on the determination of the 5-HT3E receptor gene polymorphism and expression and/or the 5-HT3C receptor gene polymorphism and expression.
  • Polymorphic alleles are typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide from the subject, using any suitable technique as is known in the art.
  • a polynucleotide is typically genomic DNA, or a polynucleotide derived from this polynucleotide, such as in a library made using ge- nomic material from the individual.
  • DNA includes genomic DNA and also cDNA.
  • obtaining a sample of DNA from the subject may comprise, e.g., obtaining genomic DNA from the subject, or obtaining RNA (in particular mRNA, e.g. total mRNA) from the subject and reversely transcribing the RNA into cDNA.
  • the presence of the polymorphism (variant) is determined in a method that comprises contacting a polynucleotide of the subject with a specific binding agent for the polymorphism (variant) and determining whether the agent binds to the polynucleotide, where the binding indicates that the polymorphism (variant) is present.
  • the binding agent may also bind to flanking nucleotides on one or both sides of the polymor- phism, for example at least 2, 5, 10, 15 or more flanking nucleotides in total or on each side.
  • the agent is able to bind the corresponding wild-type (or reference) sequence by binding the nucleotides which flank the polymorphism position, although the manner of binding will be different than the binding of a polymorphic (variant) polynucleotide, and this difference will be detectable (for example this may occur in sequence specific PCR as discussed below).
  • the presence of the polymorphism (variant) may be detected in the double stranded form, but is typically detected in the single stranded form of a polynucleotide.
  • the binding agent may be a nucleic acid, e.g., an oligo- or polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30, or more nucleotides.
  • an "oligonucleotide” generally has less than 50, 40 or 30 nucleotides.
  • the suitable length of the nucleic acid depends in particular on the capability of the nucleic acid to bind to the polymorphic (variant) sequence with higher affinity than to a different sequence (e.g., the wildtype or reference sequence). Said discrimination between the polymorphic (variant) sequence and other sequences enables the detection of the polymorphic (variant) sequence.
  • the present invention thus in particular relates to variant allele-specific oligo- or polynucleotides that hybridize to a nucleic acid molecule comprising at least one polymorphic locus, as defined herein.
  • An oligo- or polynucleotide agent which is used in the method will generally bind to the polymorphism (variant) of interest, and the flanking sequence, in a sequence specific manner (e. g. hybridize in accordance with Watson-Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymorphism (variant) and flanking region.
  • the binding agent is used as a probe.
  • the probe may be labeled or may be capable of being labeled indirectly.
  • the detection of the label may be used to detect the presence of the probe on (and hence bound to) the polynucleotide of the subject.
  • the binding of the probe to the polynucleotide may be used to immobilize either the probe or the polynucleotide (and thus to separate it from one composition or solution).
  • the polynucleotide of the subject is immobilized on a solid support and then contacted with the probe.
  • the presence of the probe im- mobilized to the solid support (via its binding to the polymorphism) is then detected, either directly by detecting a label on the probe or indirectly by contacting the probe with a moiety that binds the probe.
  • the solid support is generally made of nitrocellulose or nylon.
  • the method may be based on an ELISA system.
  • the present methods may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymorphism (variant), allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme. However the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymorphism (variant), and therefore the detection of the ligated product may be used to determine the presence of the polymorphism (variant).
  • the probe is used in a heteroduplex analysis based system to de- tect polymorphisms (variants).
  • a heteroduplex analysis based system to de- tect polymorphisms (variants).
  • the probe when the probe is bound to a polynucleotide sequence containing the polymorphism it forms a heteroduplex at the site where the polymorphism occurs (i. e. it does not form a double strand structure).
  • Such a heteroduplex structure can be detected by the use of an enzyme that is single or double strand specific.
  • the probe is an RNA probe and the enzyme used is RNAse H that cleaves the heteroduplex region, thus allowing the polymorphism to be detected by means of the detection of the cleavage products.
  • the method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3: 268-71 (1994) and Proc. Natl. Acad. Sci. 85: 4397-4401 (1998).
  • PCR reaction only if it binds a polynucleotide containing the polymorphism (i. e. a se- quence-or allele-specific PCR system).
  • a PCR product will only be produced if the polymorphism (variant) is present in the polynucleotide of the individual.
  • the presence of the polymorphism (variant) may be determined by the detection of the PCR product.
  • the region of the primer which is complementary to the polymorphism (variant) is at or near the 3' end the primer.
  • the polynucleotide the agent will bind to the wild-type or reference sequence but will not act as a primer for a PCR reaction.
  • the method may be an Restriction Fragment Length Polymorphism (RFLP) based system. This can be used if the presence of the polymorphism (variant) in the polynucleo- tide creates or destroys a restriction site that is recognized by a restriction enzyme. Thus treatment of a polynucleotide with such a polymorphism (variant) will lead to different products being produced compared to the corresponding wild-type or reference sequence.
  • RFLP Restriction Fragment Length Polymorphism
  • the presence of the polymorphism (variant) may be determined based on the change that the presence of the polymorphism (variant) makes to the mobility of the polynu- cleotide during gel electrophoresis. For instance, single-stranded conformation polymorphism (SSCP) analysis may be used. This measures the mobility of the single stranded polynucleotide on a native or non-denaturing gel compared to the corresponding wild-type or reference polynucleotide, the detection of a difference in mobility indicating the presence of the polymorphism (variant).
  • SSCP single-stranded conformation polymorphism
  • Denaturing gradient gel electropho- resis is a similar system where the polynucleotide is electrophoresed through a gel with a denaturing gradient, a difference in mobility compared to the corresponding wild-type or reference polynucleotide indicating the presence of the polymorphism (variant).
  • the presence of the polymorphism (variant) may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system. In brief, this assay uses an allele specific primer comprising the sequence around, and including, the polymorphism (variant).
  • the specific primer is labeled with a fluores- cent dye at its 5' end, a quenching agent at its 3' end and a 3' phosphate group preventing the addition of nucleotides to it. Normally the fluorescence of the dye is quenched by the quenching agent present in the same primer.
  • the allele specific primer is used in conjunction with a second primer capable of hybridizing to either allele 5' of the polymorphism (variant).
  • Taq DNA polymerase adds nucleotides to the nonspecific primer until it reaches the specific primer. It then releases polynucleotides, the fluorescent dye and quenching agent from the specific primer through its endonuclease activity. The fluorescent dye is therefore no longer in proximity to the quenching agent and fluoresces.
  • the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye is not released from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymorphism (variant) can be de- termined.
  • a polynucleotide comprising the polymorphic (variant) region is sequenced across the region which contains the polymorphism (variant) to determine the presence of the polymorphism (variant).
  • the techniques to be utilized in the present methods may be selected from DNA sequencing, sequencing by hybridization, SSCP (single strand conformational analysis), DGGE (denaturing gradient gel electrophoresis), TGGE (temperature gradient gel electrophoresis), Cleavase, Heteroduplex analysis, CMC (chemical mismatch cleavage), enzymatic mismatch cleavage, solid phase hybridization (dot blots, MASDA, reverse dot blots, oligonucleotide arrays (chips)), solution phase hybridization (Taqman, Molecular Beacons), ARMS (Amplification Refractory Mutation System), ALEX (Amplification Refractory Mutation System Linear Extension), SBCE (Single Base Chain Extension), Mini-sequencing, APEX, (Arrayed Primer Extension), RFLP (restriction fragment length polymorphism), OLA (Oligonucleotide Extension Assay) and other techniques, as is known in the
  • the present invention relates to the use of a nucleic acid selected from the group consisting of (i) a nucleic acid comprising a nucleotide sequence essentially comple- mentary or identical to the full length or to a part of the nucleotide sequence of a 5' un- translated region of a 5-HT3A receptor gene having a thymine at position -42 ; (ii) a nucleic acid comprising a nucleotide sequence essentially complementary or identical to the full length or to a part of the nucleotide sequence of a 3' untranslated region of a 5-HT3E receptor gene having an adenine at position * 76; and (iii) a nucleic acid com- prising a nucleotide sequence essentially complementary or identical to the full length or a part of the nucleotide sequence of a CDS region of a 5-HT3C receptor gene having a cytosine at position 489, for diagnosing Irritable
  • Nucleic acids are polymers of nucleotides, wherein a nucleotide comprises a base linked to a sugar which sugars are in turn linked one to another by an interceding at least bivalent molecule, such as phosphoric acid.
  • the sugar is either 2'-deoxyribose (DNA) or ribose (RNA).
  • Unnatural poly- or oligonucleotides may contain modified bases, sugars, or linking molecules, but are generally understood to mimic the complementary nature of the naturally occurring nucleic acids after which they are designed.
  • An example of an unnatural oligonucleotide is an an- tisense molecule composition that has a phosphorothiorate backbone.
  • the term "nucleic acid” as used herein also denotes polymers of nucleotides which are further modified, e.g. which comprise a label or a group capable of being labelled indirectly.
  • hybridization refers to hybridization under stringent conditions.
  • stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences.
  • Stringent conditions within the meaning of the invention, are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7 % (w/v) SDS. Nucleic acid molecules that will hybridize to a given polynucleotides under stringent conditions can be identified functionally.
  • the present invention also relates to a nucleic acid comprising a nucleotide sequence essentially complementary or identical to a part of the nucleotide sequence of the 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42, for instance, a nucleic acid comprising the sequence : 5'-gtgggcctcgtcctgagcactc-3' (SEQ ID NO:3).
  • the present invention also relates to a complement of said nucleic acid.
  • said nucleic acid or its complement is an oligonucleotide.
  • the nucleic acid is suitable to be used as a probe.
  • the present invention also relates to a nucleic acid comprising a nucleotide sequence complementary or identical to a part of the nucleotide sequence of the the nucleotide sequence of the 3' untranslated region of a 5-HT3E receptor gene having an adenine at position * 76, for instance, a nucleic acid comprising the sequence : 5'-cccctttcctaagtaccaacta-3' (SEQ ID NO:4).
  • the present invention also relates to a complement of said nucleic acid.
  • said nucleic acid or its complement is an oligonucleotide.
  • the nucleic acid is suitable to be used as a probe.
  • the present invention also relates to a nucleic acid comprising a nucleotide sequence complementary or identical to a part of the nucleotide sequence of the the nucleotide sequence of the CDS of a 5-HT3C receptor gene having a cytosine at position 489, for instance, a nucleic acid comprising the sequence : 5'-gcatctgtaacctggacatctt-3' (SEQ ID NO: 12).
  • the present invention also relates to a complement of said nucleic acid.
  • said nucleic acid or its complement is an oligonucleotide.
  • the nucleic acid is suitable to be used as a probe.
  • the present invention also relates to a kit, e.g. a predictive (patient care) test kit.
  • a test kit will aid in disease management of IBS based on the pre-determined associations between genotype and IBS.
  • Such a test could take the format of a molecular test which analyses DNA for the presence of the polymorphisms of the present invention and usually comprises (I) a nucleic acid selected from the group consisting of (i) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to a part of the nu- cleotide sequence of the 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42; (ii) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to a part of the nucleotide sequence of the 3' untranslated region of a 5-HT3E receptor gene having an adenine at position * 76; and (iii) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to a part of the nucleotide sequence of a CDS region of a 5-HT3C receptor gene having a cytosine at position 489,
  • An appropriate test kit may therefore include one or more of the following reagents or instruments: a means to detect the binding of the nucleic acid to the polymorphism, an enzyme able to act on a polynucleotide (typically a polymerase or restriction enzyme), suitable buffers for enzyme reagents, PCR primers which bind to regions flanking the polymorphism, a positive or negative control (or both), a gel electrophoresis apparatus and a means to isolate DNA from a sample.
  • the product may utilise one of the chip technologies as described by the current state of the art.
  • the test kit would include printed or machine readable instructions setting forth the correlation between the presence of the specific polymorphism and the diagnosis of IBS or likelihood that a subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a 5-HT3 receptor antagonist.
  • Certain aspects of invention are based on the finding that the increased expression of 5-HT3A and 5-HT3E subunits results in a change in 5-HT3 receptor composition and/or a higher density of 5-HT3 receptors in the epithelial cell layer of the mucosa and neurons of the enteric nervous system and therefore contributes to the pathophysiology of IBS, in particular IBS-D.
  • the present invention also relates to a method of diagnosing Irritable Bowel Syn- drome in a subject, which comprises
  • the present invention relates to a method of determining whether a subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a serotonin type 3 (5-HT3) receptor antagonist, which comprises (I) obtaining a sample from the subject; and
  • these methods of the present invention comprise determining the level of expression of both the 5-HT3A receptor gene and the 5-HT3E receptor gene.
  • the term "level of expression” is used to denote the total amount of a certain gene product, e. g. an mRNA or, preferably, a protein, which is found in a cell, tissue, tissue sample or other biological structure at the time of measurement, stan- dardized relative to a suitable quantity, e. g. the amount of a gene product known to be constitutively expressed in the tissue of interest, preferably of a housekeeping gene product, e. g. mRNA encoding ⁇ -actin or, preferably, ⁇ -actin protein, in the biological structure, or, more preferably, relative to the total content of molecules of the same class (e. g. total cellular RNA, total cellular mRNA or, preferably, total cellular protein) as the gene product of interest in the biological structure.
  • a suitable quantity e. g. the amount of a gene product known to be constitutively expressed in the tissue of interest, preferably of a housekeeping gene product, e. g. mRNA
  • constitutive expression is understood to be the expression of a gene, in particular a gene encoding a protein, that shows little, preferably no detectable, difference in expression between cells and/or tissues subjected to different stimuli and/or conditions of growth, in particular little or preferably no difference in expression between inflamed and non-inflamed tissue of the same type.
  • Determination of 5-HT3A receptor, 5-HT3C receptor and/or 5-HT3E receptor expression level in said sample may essentially be performed in situ or in vitro, or by any combination of these two possibilities applied to different portions of the sample.
  • the sample may be a body fluid, such as blood, or a tissue sample.
  • the level of expression is preferably determined on monocytes present in the body fluid. Monocytes are expected to show an enhanced level of 5- HT3A receptor gene receptor and/or 5-HT3E receptor gene expression if 5-HT3A receptor and/or 5-HT3E receptor gene expression is enhanced in the gastrointestinal tract.
  • the tissue sample is in particular a gastrointestinal tissue sample.
  • a "gastrointestinal tissue sample” is any portion of tissue taken from the subject for diagnostic purposes (biopsy), but preferably a sample of mucosa tissue, in particular colon mucosa tissue comprising enterocytes.
  • the size of the sample is chosen so as to allow for both molecular analysis and immunohistochemistry while at the same time not causing undue discomfort to the subject. More preferably, the sampling is performed using colonoscopy. Suitable procedures are known to those skilled in the art.
  • the sample is dissolved mechanically and/or chemically; in the latter case, dissolution may be performed in a suitable way to obtain the protein, in particular a 5-HT3 receptor protein comprising at least one 5-HT3A, at least one 5-HT3C receptor and/or at least one 5-HT3E subunit, in either natural (native) or denatured form, "denatured” herein being used to refer to a partial or total loss of secondary and/or tertiary structure of the protein, preferably accompanied by partial or total cloaking of the natural electric charge of the molecule by uniform non-covalent attachment of strongly charged small molecules, e. g.
  • the antibody of the invention may be able to selectively bind to a 5-HT3A, 5-HT3C and/or 5-HT3E subunit in its native form, in its denatured form, or both.
  • detergents e. g. sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • the antibody of the invention may be able to selectively bind to a 5-HT3A, 5-HT3C and/or 5-HT3E subunit in its native form, in its denatured form, or both.
  • the total proteins are separated according to their physical properties.
  • separation is preferably performed by size, using electrophoresis on an SDS-polyacrylamide gel; for native proteins, both by size and isoelectric point, using two-dimensional isoelectric focussing on a pH gradient gel.
  • the antibody against a 5-HT3A, 5-HT3C and/or 5- HT3E subunit may be directly labelled with a quantifiable tag, e. g. a fluorescent label, e. g. FITC or PE, or an enzymatically active group, e. g. peroxidase, or, preferably, it may be unlabelled.
  • a quantifiable tag e. g. a fluorescent label, e. g. FITC or PE
  • an enzymatically active group e. g. peroxidase
  • detection requires the use of a secondary antibody which is directed against the primary antibody and possesses a quantifiable tag as described above.
  • detectable groups and methods for determining them are known in the art.
  • antibody binding is followed by a suitable detection procedure, which may employ the fluorescent properties of the label or the enzymatic activities, e. g. by providing a chromogenic substrate whose conversion into a dye is then measured pho- tometrically.
  • purified 5-HT3 receptor protein comprising at least one 5-HT3A, at least one 5-HT3C and/or at least one 5-HT3E subunit, purified 5-HT3A subunit protein, 5-HT3C subunit protein or 5-HT3E subunit protein, e. g.
  • recombinantly expressed 5-HT3 receptor protein comprising at least one 5-HT3A, at least one 5- HT3C and/or at least one 5-HT3E subunit, recombinantly expressed 5-HT3A subunit protein, 5-HT3C subunit protein or 5-HT3E subunit protein, as standards allows for conversion of the physical data (fluorescence intensity, optical density, etc.) into defined amounts of protein, which may then be standardized relative to the total protein content in the sample by determining the total protein content in the latter, e. g. by a colorimetric method, e. g. Bradford's protein assay.
  • the protein content may be standardized relative to the amount of a protein known to be constitutively and constantly expressed in the sample of interest, preferably a housekeeping protein, e. g. ⁇ -actin.
  • a housekeeping protein e. g. ⁇ -actin.
  • the amount of this reference protein may be determined by methods analogous to those disclosed above. Suitable reference proteins have been described in the art.
  • proteins may be selectively precipitated from the dissolved sample using suitable antibodies, e. g. antibodies covalently linked to insolu- ble matrix materials, e. g. latex or polystyrol beads, and quantified directly, and then standardized relative to the total protein content in the sample as described above.
  • suitable antibodies e. g. antibodies covalently linked to insolu- ble matrix materials, e. g. latex or polystyrol beads, and quantified directly, and then standardized relative to the total protein content in the sample as described above.
  • immunoprecipitation is followed by electro- phoretic separation and detection with a second antibody, e. g. an antibody which rec- ognizes a 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit, more preferably an antibody which is specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5- HT3E receptor subunit.
  • a second antibody e. g. an antibody which rec- ognizes a 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit, more preferably an antibody which is specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5- HT3E receptor subunit.
  • immunoprecipitation is performed using a method selected from enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), preferably using a sandwich ELISA.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • the detection method employs two antibodies against distinct epitopes of the 5-HT3A, 5-HT3C or 5-HT3E receptor subunit, which do not steri- cally or otherwise interfere with each other's binding; one of these is attached to a substrate, preferably covalently linked to a solid matrix, and the other to a group for detection, e. g. a fluorescent or enzymatically active group.
  • the detection method comprises bringing the sample into contact with the first antibody (hereinafter referred to as “capture antibody”) attached to a substrate, washing out impurities showing low-affinity binding to the substrate, adding the labelled second antibody (hereinafter referred to as “detection antibody”) and detecting the fluorescent or enzymatically active group by a suitable method, e. g. directly by fluorometry or colorimetrically by addition of a chro- mogenic substrate which is then converted into a dye by the enzymatic activity of the label, followed by determination of optical density at a predefined wavelength.
  • a suitable method e. g. directly by fluorometry or colorimetrically by addition of a chro- mogenic substrate which is then converted into a dye by the enzymatic activity of the label, followed by determination of optical density at a predefined wavelength.
  • the detection antibody may be labelled with biotin and the detectable group cou- pled with avidin or streptavidin, thereby making it possible for more detectable groups to cluster around a single detection antibody molecule, thus achieving a stronger ampli- ficatory effect and hence a more advantageous signal : noise ratio.
  • an antibody specific for a 5-HT3A, 5- HT3C and/or 5-HT3E receptor subunit preferably specific for a 5-HT3A receptor sub- unit, a 5-HT3C receptor subunit or a 5-HT3E receptor subunit, is immobilized, by adsorption or by covalent attachment, to the inner surface of a plastic reagent vessel, preferably of a 96-well microtiter plate, so to serve as capture antibody; a tissue lysate obtained from a representative portion of tissue, preferably comprising about 1 x 10 7 cells, is solubilized in a suitable lysis buffer, preferably an approximately neutral, e. g.
  • lysis buffer comprising adequate amounts of chelators, detergents and protease inhibitors to avoid degradation of the protein(s) of interest, e. g. EDTA, Triton X- 100, sodium fluoride, urea and peptidic protease inhibitors, is brought into contact with the immobilized capture antibody; unbound protein and impurities are washed away, preferably using a balanced salt solution, more preferably phosphate-buffered saline comprising a mild detergent; a biotin-conjugated antibody selected from the group consisting of an antibody specific for a 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit, preferably specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5- HT3E receptor subunit, whose binding is not interfered with by that of the capture antibody, is applied, followed by a second round of washing as described; an streptavidin- horseradish peroxidase conjug
  • the ELISA is implemented as a protein array or microarray as described in the art. Detection of proteins bound to arrays or microarrays may be done by using fluorescent labels attached to the proteins of interest or to secondary antibodies, or by surface plasmon resonance as described in the art.
  • in situ refers to any method of detection wherein the sample is not dissolved but observed in its natural structure, thereby enabling the skilled artisan to obtain information about the spatial distribution of the protein of interest.
  • in situ detection is performed using sections, e. g. microtomic sections, of cell or tissue samples, expediently fixed and/or embedded. Suitable methods for sample preparation are well-known to those skilled in the art.
  • the sample is fixated, e. g. by treatment with glutardialdehyde, embedded into a suitable solidifying medium, e. g. microscopy grade high molecular weight polyvinyl alcohol (e. g.
  • MowiolTM a microtome
  • sections with a thickness ranging from 1 to 50 ⁇ m and more preferably from 3 to 10 ⁇ m which are then mounted upon a suitable support, e. g. a microscope slide, and sub- jected to immunohistological staining.
  • immunohistological staining is any method that allows for specific detection of distinct molecule species in a sample for optical, preferably for microscopic analysis in situ.
  • immunohistological stain- ing is performed by soaking a microscopy sample, prepared as described above, into a suitably diluted solution of a primary antibody against the structure of interest, then washing it to remove excess antibody, adding a secondary antibody which is directed against the first antibody and conjugated with a fluorescent group, washing out excess antibody once more and subjecting the stained sample to fluorescence microscopy as appropriate.
  • an "antibody” is a protein or glycoprotein molecule comprising a specific target binding domain derived from the variable regions of an immunoglobulin molecule, preferably a protein or glycoprotein molecule selected from polyclonal antibodies, e. g. polyclonal antisera; monoclonal antibodies, e. g. antibodies obtained from hybridomas; recombinant antibodies, e. g.
  • antibody fusion proteins chimeric and humanized antibodies; and fragments of polyclonal, monoclonal or recombinant antibodies, e. g. Fab and F(ab)2 fragments of the same. More preferably it is a monoclonal antibody, most preferably a monoclonal antibody of type IgG, e. g. a murine monoclonal IgG antibody.
  • the antibody may carry a covalent chemical modification, prefera- bly such as facilitates detection of the antibody, e. g. a fluorescent label.
  • Antibodies may be obtained by a variety of methods, all of which are in accordance with the present invention. Basically, any antibody with suitable binding characteristics may be used in the method of the invention.
  • a polyclonal antiserum herein understood to comprise one or more antibodies against the same target molecule but with different binding regions and potentially also with different binding epitopes on said target molecules, may be obtained by immunizing a vertebrate, preferably a mammal, more preferably a mammal selected from the group consisting of sheep, goat, horse, donkey, rabbit, rat and mouse, with the antigen or a conjugate of the antigen, e. g. the antigen covalently coupled to keyhole limpet haemo- cyanin (KLH), in the presence of a suitable adjuvant, e. g. Freund's adjuvant.
  • KLH keyhole limpet haemo- cyanin
  • the antiserum can be obtained by a method such as affinity chromatography, e. g. affinity chromatography over columns wherein a molecule with specific affinity for immunoglobulin, preferably staphylococcal Protein A or Protein G, has been immobilized on the matrix.
  • affinity chromatography e. g. affinity chromatography over columns wherein a molecule with specific affinity for immunoglobulin, preferably staphylococcal Protein A or Protein G, has been immobilized on the matrix.
  • affinity chromatography e. g. affinity chromatography over columns wherein a molecule with specific affinity for immunoglobulin, preferably staphylococcal Protein A or Protein G, has been immobilized on the matrix.
  • affinity chromatography e. g. affinity chromatography over columns wherein a molecule with specific affinity for immunoglobulin, preferably staphylococcal Protein A or Protein G, has been immobilized on the matrix.
  • the term "monoclonal antibody” means an antibody obtainable or derived from a hybridoma, e.g. an antibody secreted by a hybridoma prepared by means of hybridoma technology such as the standardized hybridoma methods according to Kohler and Milstein or any modified method based thereupon.
  • the standard method comprises immunizing an animal, preferably a rodent and more preferably a mouse, with the antigen as described above, followed by isolating the immunized animal's spleen cells, fusing them with an immortal cell line and assessing the cell fusion products for antibody production.
  • An antibody which is derived from a hybridoma and which has specificity for a target molecule of the invention, preferably for a 5-HT3A, 5- HT3C and/or 5-HT3E receptor subunit and more preferably for a 5-HT3A receptor sub- unit, a 5-HT3C receptor subunit or a 5-HT3E receptor subunit, or derivative thereof is therefore referred to as a monoclonal antibody.
  • recombinant antibody refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immu- noglobulin genes (see, for example, Taylor, L. D., et al. (1992) Nucl. Acids Res.
  • Recombinant antibodies include, for example, chimeric, CDR graft and humanized antibodies.
  • An antibody or antibody moiety of the invention may generally be produced by recom- binantly expressing the genes for light and heavy immunoglobulin chains in a host cell.
  • a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy immunoglobulin chains of said antibody, thereby expressing the light and heavy chains in the host cell and secreting them preferably into the medium in which said host cells are cultured.
  • the antibodies can be isolated from this medium. Standardized recombinant DNA methods are used in order to obtain genes for heavy and light antibody chains, e. g.
  • the general term "recombinant” refers to an artificial combination of at least two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • it refers to a method of expression wherein a nucleic acid sequence encoding the protein of interest, which in itself may be either native or recom- binant, is operably linked to functional sequences such as promoters, enhancers and/or terminators for the purpose of expression.
  • an “antibody against 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit” is an antibody whose specificity for a 5-HT3A, 5-HT3C and/or 5-HT3E receptor sub- unit is sufficiently high under the conditions of use to enable its use in a method according to the invention.
  • the antibody used is an antibody specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5-HT3E receptor subunit.
  • a “specific antibody” is an antibody which, under the conditions of use, binds to the target named but essentially does not bind to other molecules; thus, a 5- HT3A receptor subunit-specific antibody is an antibody which binds to a 5-HT3A recep- tor subunit but essentially not to non-5-HT3A receptor subunit molecules; a 5-HT3C receptor subunit-specific antibody is an antibody which binds to a 5-HT3C receptor subunit but essentially not to non-5-HT3C receptor subunit molecules; a 5-HT3E receptor subunit-specific antibody is an antibody which binds to a 5-HT3E receptor subunit but essentially not to non-5-HT3E receptor subunit molecules.
  • Antibodies specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5- HT3E receptor subunit can be obtained as described above. Further, such antibodies are known in the art. For instance, an epitope-specific anti-5-HT3A antibody, an epi- tope-specific anti-5-HT3C antibody and an epitope-specific anti-5-HT3E antibody are described in Niesler, B., Walstab, J., Combrink, S., Moller, D., Kapeller, J., Rietdorf, J., Bonisch, H., Gothert, M., Rappold, G., and Bruss, M. 2007. Characterization of the novel human serotonin receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E. MoI Pharmacol 72:8-17.
  • determining 5-HT3A receptor, 5- HT3C and/or 5-HT3E receptor gene expression level comprises detecting 5-HT3A receptor-, 5-HT3C receptor- and/or 5-HT3E receptor-mRNA, preferably by using a method which is selected from the group consisting of hybridization (Northern blotting) and RT-PCR. These methods have been described in the art.
  • the detection of 5-HT3A receptor-, 5-HT3C receptor- and/or 5-HT3E receptor-mRNA is quantitative.
  • the method of diagnosing Irritable Bowel Syndrome or the method of estimating of an individual subject's potential benefit may comprise (III) the use of a correlative function or table of any kind giving the likelihood of having Irritable Bowel Syndrome or of the benefit to be expected from treatment with a serotonin type 3 (5-HT3) receptor antagonist, preferably with a specific 5-HT3A, 5-HT3C and/or 5-HT3E receptor antagonist, for a subject with the specific level of 5-HT3A receptor, 5-HT3C receptor and/or 5-HT3E receptor expression determined in (II).
  • a serotonin type 3 5-HT3
  • E is a matrix or profile of 5-HT3A receptor, 5-HT3C receptor and/or 5-HT3E receptor relative expression in comparison to healthy controls
  • F is the correlative function incorporating previous clinical experience.
  • E is the relative overall expression of 5-HT3A receptor, 5- HT3C receptor and 5-HT3E receptor; the relative overall expression of 5-HT3A receptor and 5-HT3E receptor; the relative overall expression of 5-HT3A receptor and 5- HT3C receptor; the relative overall expression of 5-HT3C receptor and 5-HT3E receptor; the relative expression of 5-HT3A receptor; the relative expression of 5-HT3C receptor; the relative expression of 5-HT3E receptor; or any combination of the said values which have been empirically found to be useful in attributing to a given subject either a high or a low value of B, wherein a high value of B (B > 50 %) is considered as implicating a diagnosis of Irritable Bowel Syndrome or a recommendation to use 5-HT3 receptor antagonists in the treatment of a subject exhibiting such a B value.
  • E is selected from the group consisting of
  • XSA + XSE is the expression of 5-HT3x in the tissue of interest of A, where A is either a subject S having or being suspected of having IBS, in particular IBS-D, or a healthy control O not having IBS.
  • C is selected from the group consisting of [ gender ; age ; duration of disease ; abdominal pain ; discomfort ; stool frequency ; stool form (e.g. according to the Bristol Stool form scale) ; concomitant DMARD medication ; concomitant glucocorticoid medication ; concomitant antidiarrheal medication ; concomitant laxative medication] and any of its subsets, preferably any of its non-empty subsets.
  • a further aspect of the present invention is a method of treating Irritable Bowel Syndrome in a subject, which comprises administering an effective amount of a serotonin type 3 (5-HT3) receptor antagonist to the subject, wherein the subject's expression of 5-HT3A receptor and/or 5-HT3E receptor is upregulated.
  • a serotonin type 3 (5-HT3) receptor antagonist to the subject, wherein the subject's expression of 5-HT3A receptor and/or 5-HT3E receptor is upregulated.
  • Fig. 1 (A) shows pcDNA3 HTR3A-5 ' ⁇ JR constructs, not drawn to scale.
  • CMV Cytomegalovirus promoter
  • UTR untranslated region
  • CDS full-length cod- ing sequence
  • uORF upstream open reading frame
  • Fig. 3 (A) shows pRL-TK HTR3E-3 ' ⁇ JR constructs, not drawn to scale.
  • HSV TK herpes simplex virus thymine kinase promoter
  • UTR untranslated region
  • FIG. B, C show relative luciferase activity of the pRL-TK HTR3E-3 ' ⁇ JTR wild- type (c. * 76G; indicated in black) and * 76G>A (c. * 76A; indicated in grey) constructs co-expressed with 20 pmol of miR-510 or negative control mi- croRNA (neg. cont.) or anti-miR-510 precursor molecules.
  • Fig. 4 shows expression of HTR3A, HTR3E and miR-510 as detected by in situ hybridization. Neg. contr., scramble miRNA hybridization (Exiqon). 5-HT3A and 5-HT3E (both in green) subunit expression detected by immunofluores- cence.
  • Fig. 6 shows the nucleotide sequence of a 5-HT3A receptor gene including the coding region (exons only) as well as partial upstream and downstream se- quences. Nucleotide numbering is indicated at the right of the sequence, counting the A of the ATG translation initiating methionine as 1.
  • the 5- HT3A receptor protein sequence is shown below the coding DNA sequence, with numbering indicated at the right starting with 1 for the translation initiating methionine.
  • the position of introns is indicated by a vertical line, splitting the two exons.
  • the start of the first exon (transcription initiation site) is indicated by a 1 Y, the end of the last exon (poly-A addition site) by a '/'.
  • the exon number is indicated above the first nucleotide(s) of the exon.
  • the base at position -42 is underlined.
  • Fig. 7 shows the nucleotide sequence of a 5-HT3E receptor gene including the coding region (exons only) as well as partial upstream and downstream sequences. Nucleotide numbering is indicated at the right of the sequence, counting the A of the ATG translation initiating methionine as 1.
  • the 5- HT3E receptor protein sequence is shown below the coding DNA sequence, with numbering indicated at the right starting with 1 for the translation initiating methionine.
  • the position of introns is indicated by a vertical line, splitting the two exons.
  • the start of the first exon is indicated by a 1 Y
  • the end of the last exon (poly-A addition site) by a '/'.
  • the exon number is indicated above the first nucleotide(s) of the exon.
  • the base at position * 76 is underlined.
  • Fig. 8 shows the nucleotide sequence of a 5-HT3C receptor gene including the coding region (exons only) as well as partial upstream and downstream se- quences. Nucleotide numbering is indicated at the right of the sequence, counting the A of the ATG translation initiating methionine as 1.
  • the 5- HT3C receptor protein sequence is shown below the coding DNA sequence, with numbering indicated at the right starting with 1 for the translation initiating methionine.
  • the position of introns is indicated by a vertical line, splitting the two exons.
  • the start of the first exon (transcription initiation site) is indicated by a 1 Y, the end of the last exon (poly-A addition site) by a '/'.
  • the exon number is indicated above the first nucleotide(s) of the exon.
  • the base at position 489 is underlined.
  • IBS patients and healthy controls IBS patients and healthy controls.
  • the HTR3E mutation analysis was carried out on 100 patients with IBS-D (aged 18-66 years; mean age 41.5 years; 32 male), 100 IBS-C patients (aged 18-65 years; mean age 40.5 years; 5 male) and 100 healthy controls (aged 18-63 years; mean age 35.3 years; 35 male).
  • 98 IBS-D patients aged 18-66; mean age 41.7 years; 31 male
  • 99 IBS-C patients aged 18-65 years; mean age 40.6 years; 5 male
  • IBS patients were recruited from the Out Patients Departments of the University Hospitals of South Manchester (tertiary patients excluded), local general practices, advertisement in regional news papers and an existing departmental volunteer pool of patients. All satisfied the Rome Il criteria for IBS and predomi- nant Bowel habit subtype (Thompson, W. G., Longstreth, G. F., Drossman, D.A., Heaton, K. W., Irvine, E. J., and Muller-Lissner, S.A. 1999. Functional Bowel disorders and functional abdominal pain. Gut 45 Suppl 2:1143-47). All patients underwent appropriate investigations to exclude organic disease and did not show any functional disorder of the upper gastrointestinal tract that was more prominent than their IBS.
  • Genomic DNA was prepared from 5ml blood samples taken from both the patients and healthy controls using standard protocols (Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning : a laboratory manual. Cold Spring Harbor, USA: Cold Spring Harbor Laboratory Press).
  • PCR Polymerase Chain Reaction
  • Table 1 HTR3A and HTR3E primer sequences, annealing temperatures for PCR and dHPLC analysis temperatures
  • HTR3A3 ' UTRfor TCC AAT GCC AAT TCA TCT CA 421 60 exon 9 HTR3A3 ' UTRrev GAG TTT AGG GTT TCA CTG CAT TTT
  • dHPLC analysis The WAVE DNA fragment analysis system was used for the mutation analysis according to conditions recommended by the manufacturer (Transgenomic). Prior to dHPLC (denaturing high-performance liquid chromatography) analysis, the formation of heteroduplexes was achieved by denaturing the PCR products at 95 0 C for 5 min and gradually cooling them down to 4 0 C in 45 cycles (-2 °C/cycle). A 5 ⁇ l aliquot of PCR product was loaded on the DNASep column (Transgenomic). Gradient parameters and column temperatures for each amplicon were calculated using the software supplied with the WAVE system. Each amplicon was analyzed at two different column temperatures (table 1 ). In case of detection of a putative sequence variant within an amplicon, all samples were subject to direct sequencing of the respective amplicon to assure detection of homozygous variants which are not detectable using dHPLC.
  • the pcDNA3 HTR3A-5 ' ⁇ JTR wild-type and -42OT constructs were constructed by cloning the respective 5 ' UTR upstream of an existing pcDNA3 HTR3A cDNA construct.
  • To create the pRL-TK HTR3E-3 ' ⁇ JTR wild-type and * 76G>A renilla luciferase reporter constructs (c. * 76G and c.
  • the respective full-length HTR3E 3 ' UTR fragments were amplified from genomic DNA using forward primer 5 ' ATTATCTAGAG- CAGGTGC-TCACCTGCCAAC 3 ' and reverse primer 5 ' ATTATCTAG ACTG CAGAA- TTATTTATTGGG 3 ' (both with an Xbal tail).
  • the Xbal-digested PCR products were ligated into the Xbal site of the pRL-TK renilla luciferase vector (Promega).
  • Constructs were purified using the PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen) and integrity of insert sequence and orientation was verified by sequencing using the MegaBACE system (GE Healthcare).
  • HEK293 and Colo320 cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin G sodium and 100 ⁇ g/ml streptomycin sulfate. For luciferase assays, the cells were splitted into 24-well plates at approxi-
  • HEK293 cells were splitted into 75 cm cell culture flask and transfected by TranslT®-293 (Mobitec). The assay was performed 48 h after transfection.
  • Luciferase assay 400 ng (per well) of renilla luciferase reporter construct (pRL-TK HTR3E 3 ' UTR wt / * 76G>A, i.e., c. * 76G / c. * 76A) and 100 ng (per well) of reference construct pGL3-Control (firefly luciferase; Promega) were cotransfected with 5, 20 or 50 pmol of hsa-miR-510 pre-miR precursor molecules or pre-miR negative control #1 or hsa-miR-510 anti-miR miRNA inhibitor (Ambion).
  • the luciferase assay was performed using the dual-luciferase reporter assay system (Promega) and a Lucy2 lumi- nometer (Rosys Anthos Mikrosysteme) according to the manufacturers ' protocols. An aliquot of 25 ⁇ l of cell-lysate was used per luciferase activity measurement. Three replicates were performed for each transfection and luciferase activity was measured threefold.
  • HT3 receptor ligand [ H]GR65630 (86 Ci/mmol; PerkinElmer) was carried out on membranes of HEK293 cells transfected with either the pcDNA3 HTR3A-5 ' ⁇ JR wild-type or the -42C>T (C.-42C or C.-42T) construct as described previously (Niesler, B., Walstab, J., Combrink, S., Moller, D., Kapeller, J., Rietdorf, J., Bonisch, H., Gothert, M., Rap- pold, G., and Bruss, M. 2007. Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E. MoI Pharmacol 72:8-17).
  • Tissue sections preparation and in situ hybridization Six unaffected colon tissue sam- pies from four female and one male patient (55 - 78 years old; three colonic cancer patients and two patients with diverticulitis) were used for cryosections. Frozen tissue sections (12 ⁇ m) were fixed in 1x PBS containing 4 % paraformaldehyde for 20 min and then washed twice in 1x PBS for 10 min each. The sections were dehydrated and stored at - 80 0 C. Prior to hybridization, the sections were thawed and rehydrated.
  • the HTR3A and HTR3E specific hybridization probes were synthesized from 3 ' UTR cDNA fragments, subcloned into the pSTBIue-1 vector (Novagen), using the MAXIscript in vitro transcription kit (Ambion). Sense and antisense probes were generated using T7 or Sp6 polymerase. The probes were labeled with digoxigenin (DIG) by adding DIG RNA Labeling Mix (Roche) and purified with NucAway spin columns (Ambion) accord- ing to the manufacturers ' protocols.
  • DIG digoxigenin
  • DIG DIG RNA Labeling Mix
  • RNA-510 expression For detection of miRNA-510 expression, a specific 5 ' DIG-labeled antisense-locked nucleic acid (LNA) oligonucleotide (Exiqon) was used. The 5 ' DIG labeled scramble-miRNA (negative control) was purchased from the same company. MiRNA in situ hybridizations were performed according to a protocol recommended by Exiqon. Hybridization temperature for the miRCURY LNA detection probes was 56°C. The HTR3A and HTR3E specific probes were hybridized at 68°C using a modification of a previously published protocol (Ernsberger, U., Patzke, H., and Rohrer, H. 1997. The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation. Mech Dev 68:1 15-126).
  • ChAT choline acetyltransferas
  • tissue sections were washed 3 x 10 min in 1x PBS at room temperature and incubated in blocking solution containing the fluorochrome-labelled secondary antibody goat anti-rabbit Alexa fluor 488 (Invitro- gen) for three hours. From now on, every step was carried out light protected. After washing for three times for 5 min in 1x PBS, a nuclear counterstain with DAPI (1 :10.000) was carried out followed by two washes in 1x PBS. Sections were mounted in Vectashield (Vector) and stored at 4°C until microscopical investigation by a Zeiss Axiophot. Statistics.
  • neomycin transferase gene is part of the pcDNA3 vector.
  • PCR primers for HTR3E transcript amplification Myc-Tag forward, ⁇ ' -GAACAAAAACTAATATCAGAAGAAGACCTA-S ' ; HTR3E ex5/6 reverse, ⁇ ' -GGCCACATAGAACACGATC-S ' .
  • Primers for neomycin transferase gene amplification Neomycin forward, ⁇ ' -GCAGCTGTGCTCGACGTT-S ' ; Neomycin reverse, ⁇ ' -AGCCAACGCTATGTCCTGAT-S ' .
  • Example 1 Sequence variants HTR3A -42C>T (C.-42T), HTR3C 489A>C (c.489C) and HTR3E * 76G>A (c. * 76A) are associated with IBS-D.
  • HTR3A and HTR3E The 5 ' and 3 ' UTR of HTR3A and HTR3E in DNA samples of IBS-D, IBS-C patients and healthy controls were analyzed. Using dHPLC and direct sequencing of the generated PCR products, four sequence variants for HTR3A were identified. Two of these were located in the 5 ' UTR (-42C>T, -25OT) and two in the 3 ' UTR ( * 70OT, * 503C>T). For HTR3E, one variant located in the 5 ' UTR (-189G>A) and four in the 3 ' UTR ( * 76G>A, * 1 15T>G, "138OT, * 191T>C) of the gene were found (Table 2).
  • HTR3A and HTR3E identified in the mutation analysis of IBS patients and healthy controls.
  • MAF minor allele frequency in 100 control samples.
  • UCSC University of California Santa Cruz; NCBI, National Center for Biotechnology Information; dbSNP SNP database; CL, confidence interval
  • HTR3A variant -42OT and the HTR3E variant * 76G>A were found associated with the IBS-D phenotype of the disease.
  • IBS-D vs. IBS-C IBS-D vs. controls
  • IBS-D vs. non IBS-D IBS-D
  • Values indicate number of patients and healthy controls with the respective genotype.
  • Odds ratios OR
  • 95% confidence intervals CI.
  • P - values calculated using ⁇ -test [ (wt/wt) vs. (wt/-42C>T) + (-42C>T/-42C>T) ].
  • HWE Hardy-Weinberg equilibrium
  • IBS-D vs. IBS-C IBS-D vs. controls
  • IBS-D vs. non IBS-D IBS-D
  • Values indicate number of female patients and female controls with the respective genotype.
  • Odds ratios OR
  • 95% confidence intervals CI.
  • P - values calculated using Fisher's exact test [ (wt / wt) vs. (wt / *76G>A) ]
  • HTR3C CDS variant 489A>C was found associated with the IBS-D phenotype of the disease.
  • Example 2 The HTR3A -42OT (c.-42T) variant causes elevated 5-HT3A receptor density in membranes of transfected HEK293 cells.
  • radioligand binding assays using HEK293 cells transiently transfected with the pcDNA3 HTR3A 5 ' UTR wild-type or -42OT (c- 42C or c. -42T) constructs ( Figure 1A) we performed to investigate differences in bind-
  • Example 3 The HTR3E * 76G>A (c. * 76A) variant disrupts the binding site for miR-510 and significantly reduces mRNA degradation in HEK293 and Colo320 cells.
  • the HTR3E 3 ' UTR wild-type and * 76G>A luciferase constructs were co-transfected with 20 pmol of miR- 510, negative control miRNA or anti-miR-510 precursor molecules into Colo320 and HEK293 cells.
  • the anti-miR-510 precursor molecules are single-stranded RNA molecules which specifically knock-down endogenous miR-510.
  • the * 76G>A variant constructs cotransfected with miR-510 showed significantly higher (-180 %) luciferase expression compared to wild-type constructs (100 %; P ⁇ 0.001 ) in HEK293 cells, which do not endogenously express 5-HT3 receptors (Figure 3A). These findings were confirmed in 5-HT3 receptor expressing Colo320 cells ( Figure 3B). In both cell lines, no significant luciferase activity differences exist when co-transfecting negative control or anti-miR-510 precursor molecules
  • Example 4 The * 76G>A variant (c. * 76A) does not affect HTR3E mRNA levels in Colo320 cells.
  • Quantitative real-time PCR was performed to assess mRNA levels of HTR3E in Colo320 cells transfected with the pcDNA3 /-/7R3£-Myc-3 ' UTR wild-type (c. * 76G) or * 76G>A (c. * 76A) in combination with miR-510 or negative control miRNA precursor molecules.
  • No differences in the HTR3E mRNA levels (normalized to neomycin transferase mRNA levels) were detectable for any combination of transfected constructs (figure 5). This indicates that binding of miR-510 to the HTR3E mRNA does not seem to affect mRNA transcription levels or mRNA stability but decreases gene expression at the translational level.
  • Example 5 - HTR3A, HTR3E and hsa-miR-510 are co-expressed in enterocytes of the human mucosa as well as myenteric plexuses.
  • the forward primer (i) was modified at its 39 end creating the 59 part of one ACiI restriction site (59-C/CCGC-39).
  • the 39 part of the restriction site is encoded by the downstream wild-type sequence.
  • the restriction site would be destroyed. Therefore, the enzyme ACiI will cleave the PCR product of the wild-type alleles (c.-42A), but not the -42C>T variant alleles (C.-42T).

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Abstract

L'invention concerne un procédé de diagnostic et d'analyse de sous-groupe dans des sujets ayant ou étant suspectés d'avoir le syndrome du côlon irritable, des acides nucléiques et des kits et leur utilisation. L'invention porte sur un procédé précité basé sur la détection de polymorphismes dans des gènes de récepteur de sérotonine de type 3 (5-HT3). L'invention porte sur certains acides nucléiques, en particulier des sondes, et sur des kits contenant lesdits acides nucléiques. Les procédés, sondes et coffrets sont utiles pour diagnostiquer le syndrome du côlon irritable et prédire les avantages potentiels d'un traitement par un antagoniste des récepteurs 5-HT3.
PCT/EP2008/065182 2007-11-09 2008-11-07 Procédé de diagnostic et d'analyse de sous-groupe dans des sujets ayant ou étant suspectés d'avoir le syndrome du côlon irritable, acides nucléiques et kits, et leur utilisation Ceased WO2009060089A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020841A2 (fr) * 2000-09-06 2002-03-14 Glaxo Group Limited Polymorphismes du gene du recepteur de la 5-hydroxytryptamine et reponse au traitement

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020841A2 (fr) * 2000-09-06 2002-03-14 Glaxo Group Limited Polymorphismes du gene du recepteur de la 5-hydroxytryptamine et reponse au traitement

Non-Patent Citations (5)

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Title
DATABASE DBSNP [online] NCBI; 12 September 2007 (2007-09-12), CGM KYOTO: "Submitted SNP(ss) Details: ss76876582", XP002517326, retrieved from NCBI Database accession no. ss76876582 *
GERSHON ET AL: "The Serotonin Signaling System: From Basic Understanding To Drug Development for Functional GI Disorders", GASTROENTEROLOGY, ELSEVIER, PHILADELPHIA, PA, vol. 132, no. 1, 20 January 2007 (2007-01-20), pages 397 - 414, XP005920691, ISSN: 0016-5085 *
KAPELLER JOHANNES ET AL: "AGA Abstracts - W1331 Evidence for an increased expression of serotonin receptor type 3 genes in irritable bowel syndrome with diarrhea", GASTROENTEROLOGY, vol. 134, no. 4, Suppl. 1, April 2008 (2008-04-01), pages A-682, XP002517325, ISSN: 0016-5085 *
KAPELLER JOHANNES ET AL: "First evidence for an association of a functional variant in the microRNA-510 target site of the serotonin receptor-type 3E gene with diarrhea predominant irritable bowel syndrome.", HUMAN MOLECULAR GENETICS 1 OCT 2008, vol. 17, no. 19, 1 October 2008 (2008-10-01), pages 2967 - 2977, XP002517324, ISSN: 1460-2083 *
KRZYWKOWSKI K: "Do polymorphisms in the human 5-HT3 genes contribute to pathological phenotypes?", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 34, no. Part 5, November 2006 (2006-11-01), pages 872 - 876, XP002517323, ISSN: 0300-5127 *

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