WO2009060089A1

WO2009060089A1 - Method of diagnosis and subgroup analysis in subjects having or being suspected of having irritable bowel syndrome, nucleic acids and kits, and their use

Info

Publication number: WO2009060089A1
Application number: PCT/EP2008/065182
Authority: WO
Inventors: Beate Niesler; Gudrun Rappold; Johannes Kapeller
Original assignee: Universitätsklinikum Heidelberg
Priority date: 2007-11-09
Filing date: 2008-11-07
Publication date: 2009-05-14

Abstract

Method of diagnosis and subgroup analysis in subjects having or being suspected of having Irritable Bowel Syndrome, nucleic acids and kits, and their use The invention relates to a method of diagnosis and subgroup analysis in subjects having or being suspected of having IrritableBowelSyndromebased 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.

Description

Method of diagnosis and subgroup analysis in subjects having or being suspected of having Irritable Bowel Syndrome, nucleic acids and kits, and their use

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.

Irritable Bowel Syndrome (IBS) is an extremely common functional gastrointestinal disorder affecting up to 20% of the population, in which patients report abdominal pain or discomfort associated with disordered defecation or change in bowel habit. Patients can present with either constipation (IBS-C), diarrhea (IBS-D), a mixture of both (IBS- M), or unsubtyped Irritable Bowel Syndrome (IBS-U). Notably women are twice as likely to be affected as men. The condition accounts for nearly half of gastroenterology clinic referrals, markedly reduces quality of life and treatment remains far from satisfactory. The lack of adequate treatment and even unsuccessful development of new drugs stems from poor understanding of its pathophysiology. Nevertheless, there is evidence of altered gastrointestinal motility, with some studies reporting slow gastrointestinal transit and reduced motility and incidence of high amplitude propagating contractions in IBS-C. In IBS-D, accelerated transit, increased motility and high amplitude propagating contractions were described. Other studies have suggested that approximately two- thirds of IBS patients may also be more viscerally sensitive to intra-luminal events such as distension than healthy subjects, with slightly more patients with IBS-D being affected than IBS-C. Why some but not all patients exhibit these phenomenon remains unknown but evidence is emerging of a possible genetic link to these phenotypic variations. A genetic component to this disorder has also been suggested by the reports that IBS appears to aggregate within families.

Irritable Bowel Syndrome (IBS) is believed to be a multifactorial disorder. However no biochemical, radiographic, endoscopic or physiological marker has been found so far and diagnosis is accomplished primarily by observation of clinical symptoms. Also, to date, no individual susceptibility genes for IBS have been identified by either linkage or association studies.

Thus, it was an object of the present invention to provide a method of diagnosing Irritable Bowel Syndrome. Further, it was an object of the present invention to provide a method for treating 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). In the gastrointestinal (Gl) tract, 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.

To date, five human 5-HT3 receptor subunit genes have been isolated: 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. In contrast, all other subunits form functional heteromers when co-expressed with the 5-HT3A subunit (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). Expression analyses of all five subunit genes has revealed that the 5-HT3E subunit mRNA is exclusively expressed in gastrointestinal tissues like colon, small intestine and stomach while the mRNAs of other subunits are more ubiquitously expressed.

In a previous study, 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).

It has been claimed that 5-HT3 receptor antagonists were of benefit in the management of IBS-D, e.g. in nonconstipated female IBS (US 6,284,770). However, a beneficial effect was demonstrated only for a part of the subjects treated. So far, no explanation for this evident dichotomy between responders and non-responders has been pro- posed yet. Thus, it was also an object of the present invention to provide a method for subgroup analysis to individually determine whether a given subject suffering from Irritable Bowel Syndrome may potentially benefit from the administration of a 5-HT3 receptor antago- nist.

Surprisingly, it was found that certain polymorphisms in the 5-HT3A, 5-HT3C and 5- HT3E receptor genes are associated with Irritable Bowel Syndrome, in particular diarrhea-predominant Irritable Bowel Syndrome.

More specifically, mutation analysis was carried out in 200 patients with Irritable Bowel Syndrome and 100 healthy controls. The HTR3A 5^'UTR variant -42OT and the HTR3E 3^'UTR variant ^*76G>A (the c.^*76A allele) 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) can be used to downregulate increased 5-HT3 levels.

Thus, the present invention relates to a method of diagnosing Irritable Bowel Syndrome in a subject, 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 (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. the sequence of the more frequent allele) at position -42 in the 5' untranslated region of the 5-HT3A receptor gene (the so-called HTR3A 5'UTR variant -42OT; also referred to as HTR3A C.-42T) and 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). These single nucleotide polymorphisms (SNPs) are identified by the change in nucleotide and the position of the polymorphism; the numbering of nucleotides is that of Fig. 6 for the 5-HT3A receptor gene, Fig. 7 for the 5-HT3E receptor gene, and Fig. 8. for the 5-HT3C receptor gene.

Preferably, 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. According to a particular embodiment, methods of the present invention comprise determining the genotype of the subject at all three polymorphic allelic sites.

As used herein, a "5-HT3A receptor gene" is gene comprising a nucleic acid sequence with an overall sequence identity of at least 70 %, preferably at least 80 %, more pref- erably at least 90 % or at least 95 % and most preferably at least 99 % with the human 5-HT3A receptor gene, e.g., the gene having the sequence as laid down in SEQ ID NO:1 (HTR3A genomic hg18_knownGene_NM_000869 range=chr11 :113351 120- 1 13366242; GenelD: 3359 (ENTREZ)) or as depicted in Figure 6.

As used herein, a "5-HT3E receptor gene" is gene comprising a nucleic 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 gene, e.g., the gene having the sequence as laid down in SEQ ID NO:2 (HTR3E genomic hg18_knownGene_NM_182589 range=chr3:185300661- 185307476; GenelD: 285242 (ENTREZ)) or as depicted in Figure 7.

As used herein, a "5-HT3C receptor gene" is gene comprising a nucleic acid sequence with an overall sequence identity of at least 70 %, preferably at least 80 %, more pref- erably at least 90 % or at least 95 % and most preferably at least 99 % with the human 5-HT3C receptor gene, e.g., the gene having the sequence as laid down in SEQ ID NO:10 (HTR3C genomic hg18_knownGene_NM_130770 range=chr3: 185253529- 185261 153; GenelD: 170572 (ENTREZ)) or as depicted in Figure 8.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "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 polynucleotide comparison include the following: (1 ) Algorithm: Needleman and Wunsch, J. MoI Biol. 48:443-453 (1970) Comparison matrix: matches=+10, mismatch=O Gap Penalty: 50 Gap Length Penalty: 3. Preferred parameters for polypeptide sequence comparison include the following:(1 ) Algorithm: Needleman and Wunsch, J. MoI Biol. 48:443-453 (1970) Comparison ma- trix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci, USA. 89:10915- 10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4.

As used herein, "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). As is well known in the art, 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.

As used herein, a "genetic subset" of a population consists of those members of the population having a particular genotype. In the case of a biallelic polymorphism, a population can potentially be divided into three subsets: homozygous for allele 1 , heterozygous, and homozygous for allele 2.

The presence of one polymorphism (variant), i.e., of the HTR3A 5'UTR variant -42OT (i.e., the C.-42T allele) or the HTR3E 3'UTR variant ^*76G>A (i.e., the c.^*76A allele) or the HTR3C CDS variant 489A>C (i.e., the C.489C allele), or the presence of two polymorphisms (variants), 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 polymorphism (variant)-exhibiting subgroup of patients where it can be expected to be effective. In subjects which are homozygous for at least one, at least two or all of said variants 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.

As used herein, 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. In particular, as used herein, 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).

The concept of "quality-adjusted life years" and "disability-adjusted life years" is used extensively to evaluate drugs and treatments, in particular in the context of those diseases where morbidity and disability are more of a concern than mortality. Essentially, each year the life time following treatment is multiplied with an index factor which ranges from 1.0 to indicate perfect quality of life, or zero disability, to 0.0 to indicate death, or complete disability, and the sum of these products is compared to the value obtainable without treatment. Suitable definitions and methods for determining gains and losses in QALYs and DALYs have been described in the art.

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.

Being capable of determining the subject's potential benefit from treatment with 5-HT3 receptor antagonists has several advantages.

First, 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. Second, many existing 5-HT3 receptor antagonists, such as alosetron, are burdened with a propensity for severe side effects. Thus, 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.

Because of the aforesaid pleiotropic effects and the unwanted side effects likely to re- suit from them, such as constipation and ischemic colitis, it is advantageous to distinguish likely responders from likely non-responders before the initiation of treatment with 5-HT3 receptor antagonists. Hence, one advantage of the present invention is to be able to restrict 5-HT3 receptor antagonist treatment to cases of verifiable need. As used herein, 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.

The term "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. According to one embodiment of the invention, a 5-HT3 receptor comprises at least one 5-HT3A receptor subunit. According to a further embodiment of the invention, a 5-HT3 receptor comprises at least one 5- HT3E receptor subunit. According to a further embodiment of the invention, a 5-HT3 receptor comprises at least one 5-HT3C 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-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.

As used herein, 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.

As used herein, 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.

As used herein, 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.

Preferably, 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.

Efficient 5-HT3 receptor antagonist and methods of synthesizing them have been de- scribed in the art, e. g., in

WO 05/082887 (pyrimidine derivatives);

WO 04/045612 (imidazopyridines);

WO 04/094418 (imidazopyridines);

WO 03/061657; WO 01/098253 (1-amino-alkylcyclohexanes);

WO 98/024790 (1 ,4-diazabicyclo(2.2.2)oct-2-ylmethyl derivatives);

WO 95/032209 (azabicycloalkyl derivatives of imidazo[1 ,5-a]indol-3-one;

WO 95/032208 (imidazolylalkyl derivatives of imidazol(5,1-c)(1 ,4)benzoxazin-1-ones);

WO 95/032204 (imidazolylalkyl derivatives of imidazo(1 ,5-a)indol-3-ones); WO 95/01 1245 (indole derivatives);

WO 95/009167 (indolines)

WO 94/022862 (indolizine derivatives);

WO 94/013667 (azanoradamantanes);

WO 94/012494 (dimethylbenzofurans and dimethylbenzopyrans); WO 94/01 1347 (phenyl imidazolidinone derivatives);

WO 94/002482 (1-azaadamantane derivatives);

WO 94/000454 (benzimidazoles);

WO 94/000449 (benzimidazole compounds);

WO 93/025555 (imidazo[5,1-c][1 , 4]benzoxacin-1-ones) WO 93/018025 (N,N'-disubstituted amide derivatives);

WO 93/017019 (2,6-methano-2H-quinolizin derivatives);

WO 93/008186 (pyridine-3-carboxylic acid esters or amides);

WO 93/008185 (N-aryl-N1-azabicyclo-ureas);

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 92/012149 (azabicyclic and azatricyclic derivatives);

WO 92/009284 (2,6-methano-2H-1-benzoxocincarboxamides);

WO 91/017161 (isochinoline amides and esters); WO 91/007402 (azabicyclo amides and esters);

WO 91/005783 (heterocyclic compounds);

WO 91/001316 (9-azabicyclo[3.3.1]nonane-derivatives);

WO 90/006309 (heteroazabenzobicyclic carboxamides);

WO 89/009217; EP-A 874 833 (4,5-dihydronaphth[1 ,2-c]isoxazoles);

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);

(see also Andrew J Thompson and Sarah CR Lummis, Expert Opin. Ther. Targets:

2007 April; 1 1 (4): 527-540).

According to a particular embodiment, 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.

According to a further embodiment, the 5-HT3 receptor antagonist is an antibody that binds to the 5-HT3 receptor.

According to a further particular embodiment, 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. Preferably, 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.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a 5-HT3A, 5-HT3C or 5- HT3E polynucleotide. 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. Preferably, 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. These modifications can be internal or at one or both ends of the antisense molecule. For example, 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.

According to a further particular embodiment, 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. For example, 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. The 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. Alternatively, if it is desired that the cells stably retain the DNA construct, 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.

According to a further particular embodiment, 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).

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.

There are several approaches for designing suitable molecules capable of mediating RNA interference. For instance, 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.

Further, 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). Still further, 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).

Basically, any pharmacologically acceptable 5-HT3 receptor antagonist may be used in the treatment of Irritable Bowel Syndrome.

However, according to the present invention, preference is given to 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.

According to a further particular embodiment, 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. Further, the miRNAs disclosed above are examples of 5-HT3E receptor subunit- specific antagonists.

As used herein, "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 ).

In a preferred embodiment of the invention, 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. According to a particular embodiment, 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. Such 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. Thus, 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.

Typically 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.

In one embodiment 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. On the other hand, 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.

In one embodiment of the present methods 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).

In another embodiment of the invention 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. In the case of a protein polymorphism 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).

In one embodiment the probe is used in a heteroduplex analysis based system to de- tect polymorphisms (variants). In such a system 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. Typically 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).

In one embodiment the polynucleotide agent is able to act as a primer for a

PCR reaction only if it binds a polynucleotide containing the polymorphism (i. e. a se- quence-or allele-specific PCR system). Thus a PCR product will only be produced if the polymorphism (variant) is present in the polynucleotide of the individual. Thus the presence of the polymorphism (variant) may be determined by the detection of the PCR product. Preferably the region of the primer which is complementary to the polymorphism (variant) is at or near the 3' end the primer. In one embodiment of this system 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.

Thus the detection of the presence of particular restriction digest products can be used to determine the presence of the 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). Denaturing gradient gel electropho- resis (DGGE) 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).

In the assay, when the allele comprising the polymorphism (variant) is present 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. In the presence of the allele which does not comprise the polymorphism (variant) 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.

In another method of detecting the polymorphism (variant) 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).

Accordingly, 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 art.

Thus, 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 Bowel Syndrome or determining whether a subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a serotonin type 3 receptor (5-HT3) antagonist. In each case, the nucleic acid will comprise a nucleotide sequence essentially complementary or identical to a gene portion which portion comprises the polymorphic locus and allows for appropriate hybridization of the nucleic acid to said portion.

"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. In naturally occurring nucleic acids, 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.

The term "essentially" is meant to denote a degree of complementarity or identity that is sufficient to allow hybridization under the conditions of the experiment.

Unless indicated otherwise, hybridization refers to hybridization under stringent conditions.

The term "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. According to a particular embodiment, said nucleic acid or its complement is an oligonucleotide. According to a further particular embodiment, 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 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 kit, e.g. a predictive (patient care) test kit. Such 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, such as the nucleic acids disclosed herein; and (II) a means to determine whether the nucleic acid binds to a sample of DNA from a subject.

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.

Thus, the present invention also relates to a method of diagnosing Irritable Bowel Syn- drome in a subject, which comprises

(I) obtaining a sample from the subject; and

(II) determining whether the level of expression of a 5-HT3A receptor and/or 5-HT3E receptor gene is upregulated in the sample, the upregulation of the level of expression indicating that the subject has Irritable Bowel Syndrome or may be at risk of developing Irritable Bowel Syndrome.

Further, 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

(II) determining whether the level of expression of a 5-HT3A recepto and/or 5-HT3E receptor gene is upregulated in the sample, the upregulation of the level of expression indicating the subject's potential benefit. Preferably, 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. An elevated expression of one receptor gene, i.e. of the 5-HT3A receptor gene or the 5-HT3E receptor gene, or of both receptor genes, indicates that the subject is likely having Irri- table Bowel Syndrome and/or the subject's potential benefit.

As used herein, 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 α-actin or, preferably, α-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.

As used herein, "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.

In case of a body fluid, 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. As used herein, 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. Preferably, 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.

For in vitro determination of 5-HT3A, 5-HT3C and 5-HT3E receptor gene expression levels, 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. by boiling with detergents, e. g. sodium dodecyl sulfate (SDS), under reducing conditions, e. g. in the presence of β-mercaptoethanol. 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.

Expediently, after dissolution the total proteins are separated according to their physical properties. For denatured proteins, 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. These techniques are familiar to the person skilled in the art.

Subsequently, the separated proteins are subjected to detection using an antibody of the invention as primary antibody. 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. In the latter case, detection requires the use of a secondary antibody which is directed against the primary antibody and possesses a quantifiable tag as described above. Such detectable groups and methods for determining them are known in the art.

In both cases, 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.

The use of defined amounts of 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. Alternatively, and more expediently for all-immunology methods such as protein arrays, 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. α-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.

Alternatively, for in vitro determination 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.

In a particular embodiment of the invention, 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.

In another particularly preferred embodiment of the invention, immunoprecipitation is performed using a method selected from enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), preferably using a sandwich ELISA.

It is especially preferred that 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. Alterna- tively, 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.

In a most preferred embodiment of the invention, 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 10⁷ cells, is solubilized in a suitable lysis buffer, preferably an approximately neutral, e. g. pH 7 - 7.5, 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 conjugate is added, followed by a third round of washing as described; a substrate solution comprising a chromogenic substrate for horseradish peroxidase, preferably a solution comprising H₂O₂ and tetramethylbenzidine, is applied and incubated for a predetermined period of time prior to termination of the reaction by addition of acid, preferably concentrated sulfuric acid; optical density is measured, preferably at λ = 450 nm; and the amount of 5-HT3A and/or 5-HT3E receptor subunit is determined by comparison with internal standards with defined amounts of the same, e. g. recombinantly expressed and purified.

In another particularly preferred embodiment of the invention, 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.

All of the aforementioned in vitro methods will finally yield the total 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit content (w/w) of the sample. These values are then used as part of the matrix or profile E to estimate, by correlation with previous clinical experience embodied in a function F as described in formula (1 ) and (2) below, the subject's potential benefits.

As used herein, "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. Preferably, 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. In a preferred embodiment of the invention, 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. Mowiol™), and cut into a series of sections using a microtome, preferably sections with a thickness ranging from 1 to 50 μm and more preferably from 3 to 10 μm, which are then mounted upon a suitable support, e. g. a microscope slide, and sub- jected to immunohistological staining.

As used herein, "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. In a preferred embodiment of the invention, 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. By using primary antibodies derived from different species and suitable secondary antibodies with distinct labels, more than one target can be assessed in parallel, and colocalization of different structures can be determined, e. g. the presence of a 5-HT3A, 5-HT3C and/or 5-HT3E receptor subunit. Materials and procedures applicable to this task are known to those skilled in the art. It is particularly preferable that analysis of the microscopic images is performed using automatic evaluation software which allows for suitable quantifications of the micrographs. The results thus obtained, e. g. a two-dimensional matrix giving a map of the distribution of expression values within a cell or tissue, are useful as components of the expression matrix or profile E in formula (1 ) or (2) as described below, once their clinical significance has been quanti- fied.

Preferably, both approaches are combined to obtain a complete set of 5-HT3A, 5- HT3C and/or 5-HT3E receptor subunit expression data, with a part of the sample being dissolved for in vitro determination and the rest for in situ measurements. As used herein, 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. Suitable procedures and formulations for immunization have been described extensively in the art. Following bleeding of the animals and removal of the particulate blood components such as blood cells by a suitable method, e. g. centrifugation, 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. Such methods are well-known to those skilled in the art, as are methods for determining pu- rity, e. g. electrophoresis, and affinity, e. g. sorbent assays such as RIA or ELISA.

As used herein, 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. Essentially, 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.

As used herein, the term "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.

20:6287-6295); or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. 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. In order to recombinantly express an antibody, 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. by deriving them by RT-PCR from the genes expressed in a particular hybridoma cell, to insert said genes into recombinant expression vectors and to introduce said vectors into host cells. Methods of this kind are described, for example, in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989) (hereinafter referred to as "Sambrook"), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in US 4,816,397 by Boss et al..

Standard recombinant DNA and molecular cloning techniques used herein are well- known in the art and are also described more fully in Sambrook.

As used herein, 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. In particular, 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.

As used herein, 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.

It is particularly preferred that the antibody used is an antibody specific for a 5-HT3A receptor subunit, a 5-HT3C receptor subunit or a 5-HT3E receptor subunit.

As used herein, 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.

In a further preferred embodiment of the invention, 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.

In a particular embodiment of the invention, 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). Expediently, the function or table will reflect experience from previous clinical trials.

Generally, the function or table will take the form

B = F( E ) (1 ), wherein

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,

B is the probability of beneficial effects, and

F is the correlative function incorporating previous clinical experience.

It will be appreciated that F does not have to be an arithmetic function but may be a plain list of clinical findings. 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.

According a more particular embodiment, the function or table will take the general form B = F( E, C ) (2), where B, F and E have the meaning as described above and C is a matrix or profile of additional relevant characteristics of the individual subject, such as further individual and/or clinical parameters, concomitant treatment and the like. It is to be expected that this will allow for more reliable estimation of the likelihood of benefits from treatment, once the required clinical studies have been completed.

Thus, in a preferred embodiment of the invention E is selected from the group consisting of

[ XSA / XOA ] ; [ XSC / XOC ] ; [ XSE / XOE ] ;

[ (XSA + XSE) / (X0A+X0E) ] ; [ (XSA + XSC) / (XOA+XOC) ] ; [ (XSC + XSE) / (XOC+XOE) ] ; and [ (XSA + XSC + XSE) / (XOA+XOC+XOE) ] ; wherein XAx 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.

In a preferred embodiment of the invention, 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.

The present invention will now be described in more detail with reference to the following examples and drawings which illustrate the invention without limiting it in any respect.

In the figures,

Fig. 1 (A) shows pcDNA3 HTR3A-5^'ϋJR constructs, not drawn to scale. CMV, Cytomegalovirus promoter; UTR, untranslated region; CDS, full-length cod- ing sequence; uORF, upstream open reading frame; and

(B) shows maximum [3H]GR65630 binding (Bmax) to membranes of HEK293 cells transfected with pcDNA3 HTR3A-5^'UTR wild-type or -42C>T (C.-42C or c. -42T) constructs. Values are means of 5 experiments ± SEM (n = 5). ^*, P < 0.05.

Fig. 2 (A) shows Predicted miR-510 binding site in the wild-type (wt) 3^'UTR of HTR3E and location of the ^*76G>A variant (c.^*76G and c.^*76A), and (B) shows relative luciferase activity of the pRL-TK HTR3E- 3^'UTR wild- type construct (c.^*76G), co-expressed with different amounts of miR-510 (indicated in black) or negative control miRNA (neg. cont; indicated in gray) in Colo320 cells. Renilla luciferase (pRL-TK) activity was normalized to firefly luciferase (pGL3-Control). Values are means ± SEM for three transfec- tions (n = 3). ^**, P < 0.001 (miR-510 vs. negative control).

Fig. 3 (A) shows pRL-TK HTR3E-3^'ϋJR constructs, not drawn to scale. HSV TK, herpes simplex virus thymine kinase promoter; UTR, untranslated region, and

(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. Assay was performed in HEK293 (B) and Colo320 (C) cells. Renilla luciferase (pRL-TK) activity was normalized to firefly luciferase (pGL3 control). Values are means ± SEM for three transfections each measured threefold (n = 9). ^**, P < 0.001 {HTR3E 3^'UTR wt vs. ^*76G>A, i.e. c.^*76G vs. c.^*76A).

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. 5 shows relative HTR3E mRNA concentration in Colo320 cells co-transfected with full-length HTR3E 3^'UTR wild-type (wt) or ^*76G>A construct (c.^*76G or c.^*76A) and miRNA-510 or negative control miRNA (neg. cont.) normalized to Neomycin-transferase mRNA concentration. Values are means ± SEM for two independent experiments with double transfections (n = 4).

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 ¹Y, 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 (transcription initiation site) is indicated by a ¹Y, 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 ¹Y, 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.

EXAMPLES

Methods

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). For the HTR3A mutational analysis, 98 IBS-D patients (aged 18-66; mean age 41.7 years; 31 male) and 99 IBS-C patients (aged 18-65 years; mean age 40.6 years; 5 male) of the same patient pools and the 100 healthy controls were screened. IBS patients with a mixed Bowel habit (IBS-M) were excluded from the study. 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. In addi- tion, no subject had a history of major psychiatric disorder or history of alcohol or substance abuse. Healthy controls were recruited by advertisement. All subjects were Caucasian and drank below the recommended safe alcohol limit (<21 units/week), smoked < 5 cigarettes per day, and had not participated in a clinical trial of any drug within the previous 30 days. Written consent was obtained from all subjects and the study was approved by the South Manchester Medical Research Ethics Committee.

Preparation of genomic DNA. 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).

Polymerase Chain Reaction (PCR). PCRs were performed in 25 μl volumes containing 50 ng of genomic DNA as template, 10 pmol of each primer, 200 μM dNTPs (MBI Fer- mentas), 2 mM MgSO , 10 mM KCI, 10 mM (NH ) SO , 20 mM Tris-HCI, 0.1 % Triton

4 4 2 4 X-100 and 1.25 U of Taq DNA Polymerase (NEB). Thermal cycling was performed in a PTC-200 (MJ Research) or Mastercycler gradient thermal cycler (Eppendorf). Annealing temperatures (T ) and sequences of the UTR specific HTR3A and HTR3E primers and the HTR3C exon 5 primers are shown in table 1. Cycling conditions were: Initial denaturation at 94 ⁰C for 2 min followed by 35 cycles of 94 ⁰C for 30 s, T for 30 s and

A 72 ⁰C for 30 s. The final extension step was at 72 ⁰C for 5 min. A 3 μl aliquot of each PCR product was analyzed on a 1.5 % agarose gel.

Table 1 : HTR3A and HTR3E primer sequences, annealing temperatures for PCR and dHPLC analysis temperatures

amplicon primer primer sequence (5^' → 3^') size TA dHPLC

(bp) ("C) ("C)

HTR3A

5^'UTR HTR3Aex1 infer TAC TCC TTG GGG AAA CAT GG 483 60 63.5 exon 1 HTR3Aex1 inrev CCT CGG AGG ACT GAA GCA T 64.0

3^'UTR - 1 HTR3Aex9infor GAA CCA TGT TCA GGT CAC CA 550 60 62.5 exon 9 HTR3Aex9inrev TTT GGT GGA AGG GTT CAG AC 64.0

3^'UTR - 2 HTR3A3^'UTRfor TCC AAT GCC AAT TCA TCT CA 421 60 exon 9 HTR3A3^'UTRrev GAG TTT AGG GTT TCA CTG CAT TTT

HTR3E

5 UTR HTR3Eex1 infer GGA CGT ATA GCA CAG CAG A 260 58 57.0 exon 1 HTR3Eex1erev CGC CCG TGA TAA AAT GAA AG 57.5

3^'UTR HTR3Eex8efor CGT CAT ATG CCT CTG GAA CA 397 68/64/60 58.5 exon 8 HTR3Eex8inrev ATA GGC GTG AAC CAC TGC AC (SD) 59.1

HTR3C

CDS HTR3Cex5efor AAA GAG CCC AGA AGG AGA GC 364 60 exon 5 HTR3Cex5inrev TGG AGC AAC ACT GAT CCA AA

For the analysis of HTR3A 3^'UTR - 2 and HTR3C exon 5, only direct sequencing was performed. UTR, untranslated region; bp, base pairs; TA, annealing temperature; SD, step down PCR; dHPLC, denaturing high performance liquid chromatography.

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 ⁰C for 5 min and gradually cooling them down to 4 ⁰C in 45 cycles (-2 °C/cycle). A 5 μl 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.

Purification and direct sequencing of PCR products. A 5 μl aliquot of PCR product was treated with 2 U shrimp alkaline phosphatase (SAP) and 5 U exonuclease I (Exol; MBI Fermentas) for 15 min at 37 ⁰C followed by inactivation at 80 ⁰C for 15 min. Two μl of the Exol/SAP-treated PCR product was used for direct sequencing using the DYE- namic ET Terminator Cycle Sequencing Kit according to the manufacturer's protocol (GE Healthcare). The MegaBACE 1000 sequencer and the software provided by the manufacturer (GE Healthcare) were used for analysis of the sequence reaction products. Expression and luciferase reporter constructs. The pcDNA3 HTR3A-5^'\JTR wild-type and -42OT constructs (c.-42C and C.-42T; Figure 1A) 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.^*76A; Figure 3A), 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).

Cell culture and transfection. HEK293 and Colo320 cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. For luciferase assays, the cells were splitted into 24-well plates at approxi-

5 mately 5.0 x 10 cells per well prior to transfection. For luciferase assays, cells were transiently transfected using 4 μg of polyethylenimine (PEI; Sigma-Aldrich) per 1 μg of construct DNA and cells were harvested 24 h after transfection. For radioligand binding

2 assays, 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 μl of cell-lysate was used per luciferase activity measurement. Three replicates were performed for each transfection and luciferase activity was measured threefold.

Membrane preparation and radioligand binding assay. Radioligand binding with the 5-

3 HT3 receptor ligand [ H]GR65630 (86 Ci/mmol; PerkinElmer) was carried out on membranes of HEK293 cells transfected with either the pcDNA3 HTR3A-5^'ϋJR 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 μm) 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 ⁰C. 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. 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).

Immunofluorescence. For localization of 5-HT3A and 5-HT3E subunits in human colon tissue sections, immunofluorescence experiments were carried out as follows : tissue sections (8 μm) were fixed by incubation in 4 % paraformaldehyde for 20 min. Afterwards they were washed three times for 10 min in 1x PBS at room temperature. Then slides were blocked in 4 % goat serum / 0.25 % Triton-X-100 / PBS. The first antibodies rabbit anti-5-HT3A or anti-5-HT3DE were diluted 1 : 100 in blocking solution and applied over night at room temperature. Afterwards, 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. Comparison of genotype frequencies, association analyses and test for deviation from the Hardy-Weinberg equilibrium were performed using an online tool provided by the Institute of Human Genetics in Munich (http://ihg.gsf.de/cgi- bin/hw/hwa1.pl) and the Fisher^'s Exact Test (http://www.matforsk.no/ola/fisher.htm). For the luciferase and radioligand binding assay results, the independent samples t- test was performed using the MedCalc software (http://www.medcalc.be).

In silico analysis of microRNA binding sites. We performed in silico analysis of micro- RNA binding sites, using the miRBase Target database (http://microrna.sanger.ac.uk) (Griffith s-J ones, S., Grocock, R. J., van Dongen, S., Bateman, A., and Enright, AJ.

2006. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140-144).

Real-time PCR. The pcDNA3 HTR3E-Myc-3^'UTR wild-type and ^*76G>A (c.^*76G and c.^*76A) expression constructs were created by cloning the respective full-length 3^'UTR downstream of the HTR3E coding sequence of a previously described pcDNA3 HTR3E Myc-epitope tagged construct (11 ). Colo320 cells were cotransfected with 10 μg (per 10 cm dish) of pcDNA3 H7R3£-Myc-3^'UTR wild-type or ^*76G>A (c.^*76G or c.^*76A) construct and 400 pmol of hsa-miR-510 pre-miR precursor molecules (Ambion). Cells were harvested 24 h after transfection and total RNA was isolated using the RNeasy Mini Kit (Qiagen). DNase treatment was performed. 1 μg of purified total RNA was reverse transcribed using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. Quantitative real-time PCR was performed using ABsolute QPCR SYBR Green Capillary Mix (Abgene Limited) and the LightCycler 2.0 system according to protocols (Roche). HTR3E expression levels were normalized to neomycin transferase gene expression levels. The neomycin transferase gene is part of the pcDNA3 vector. PCR primers for HTR3E transcript amplification: Myc-Tag forward, δ^'-GAACAAAAACTAATATCAGAAGAAGACCTA-S^'; HTR3E ex5/6 reverse, δ^'-GGCCACATAGAACACGATC-S^'. Primers for neomycin transferase gene amplification: Neomycin forward, δ^'-GCAGCTGTGCTCGACGTT-S^'; Neomycin reverse, δ^'-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.

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).

Table 2

HTR3A and HTR3E sequence variants

Gene Location Sequence ^■> UCSC NCBI MAF (CI.) dbSNP

HTR3A 5' UTR (exon l) -42OT 113.351.216 rs 1062613 0.150 (0.104

0.207)

-25OT 113.351.233 rs62625041 0.000 (0.000

0.018)

3' UTR (exon 9) *70OA 113.365.765 rs62625042 0.005 (0.000

0.028) *503OT 113.366.198 rs62625043 0.040 (0.017

0.077)

HTR3E 5' UTR (exon l) -189 A>G 185.300.666 rs34611203 0.185 (0.134

0.246)

3' UTR (exon 8) *76G>A 185.307.251 rs62625044 0.030 (0.011

0.064)

*115T>G 185.307.290 rs62625045 0.005 (0.000

0.028)

*138OT 185.307.313 rs62625046 0.000 (0.000

0.018)

*191T>C 185.307.366 rs62621663 0.000 (0.000

0.018)

Sequence variants of 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

Statistical analyses on the genotype frequencies obtained for the identified variants of both genes were performed to determine whether there are significant differences between the two IBS subgroups and healthy controls. The HTR3A variant -42OT and the HTR3E variant ^*76G>A were found associated with the IBS-D phenotype of the disease.

The heterozygous and the homozygous genotype of the HTR3A -42OT (c.-42T) variant was significantly more frequent in IBS-D compared with both the healthy control (P = 0.020; odds ratio (OR) = 2.01 ) and IBS-C subgroups (P = 0.034; OR = 1.89) or compared with a pooled group of non IBS-D individuals (IBS-C and controls; P = 0.009; OR = 1.95). There were no genotype frequency differences in IBS-C patients compared to healthy controls (Table 3). Table 3

HTR3A -42OT genotype frequencies in IBS patients and healthy controls

Genotype IBS-D IBS-C Controls Non IBS-D

(n = 98) (n = 99) (n = 100) (IBS-C + controls) wt / wt 55 70 72 142 wt / -42OT 35 23 26 49

-42OT / -42OT 8 6 2 8

P = 0.034 P = 0.020 P = 0.009

OR = 1.89 OR = 2.01 OR = 1.95

C.I. = [1.05 - 3.40 ] CL = [LI l - 3.63 ] C.I. = [1.18 - 3.22 ]

(IBS-D vs. IBS-C) (IBS-D vs. controls) (IBS-D vs. non IBS-D)

Values indicate number of patients and healthy controls with the respective genotype.

Odds ratios (OR), 95% confidence intervals (CI.) and P - values calculated using χ -test [ (wt/wt) vs. (wt/-42C>T) + (-42C>T/-42C>T) ].

No deviation from the Hardy-Weinberg equilibrium (HWE) was present in the IBS- subgroups or in the control group for the -42OT genotypes.

The heterozygous genotype of the HTR3E ^*76G>A (c.^*76A) variant was more frequent in female IBS-D patients compared to female healthy controls (P = 0.033; OR = 8.53) or female IBS-C patients (P = 0.054; OR = 4.04) or compared to the pooled group of non IBS-D females (P = 0.008; OR = 5.17), while there were no genotype frequency differences between female IBS-C patients and female healthy controls (Table 4).

Table 4

HTR3E *76G>A genotype frequencies in female IBS patients and female healthy controls

Genotype IBS-D IBS-C Controls Non IBS-D

(n = 68) (n = 95) (n = 65) (IBS-C + controls) wt / wt 60 91 64 155 wt / *76G>A 8 3 1 4

*76G>A / *76G>A 0 1 0 1

P = 0.054 P = 0.033 P = 0.008

OR = 4.04 OR = 8.53 OR = 5.17

CL = 1.03 - 15.86 CL = 1.04 - 70.28 CL = [ 1.50 - 17.80 ]

(IBS-D vs. IBS-C) (IBS-D vs. controls) (IBS-D vs. non IBS-D)

Values indicate number of female patients and female controls with the respective genotype.

Odds ratios (OR), 95% confidence intervals (CI.) and P - values calculated using Fisher's exact test [ (wt / wt) vs. (wt / *76G>A) ]

For the HTR3E ^*76G>A variant, no deviation from HWE was detected in the IBS patients or the healthy controls. Further results on the association of said variants with IBS-D are found in Kapeller J, Houghton LA, Monnikes H, Walstab J, Moller D,Bόnisch H, Burwinkel B, Niesler B, 2008, 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, 17 (19), 2967-2977, which is incorporated herein in its entirety by reference.

Further, using dHPLC and direct sequencing of the generated PCR products and statistical analyses as described above the HTR3C CDS variant 489A>C (c.489C) was found associated with the IBS-D phenotype of the disease.

The homozygous genotype of the HTR3C 489A>C (c.489C) variant was more frequent in female IBS-D patients compared to female healthy controls (P = 0.0019; OR = 4.98) or compared to the pooled group of non IBS-D females (P = 0.0086; OR = 3.23) (Table 6).

Table 6

HTR3C C.498OA genotype frequencies in female IBS patients and female healthy controls

Genotype IBS-D IBS-C Controls Non IBS-D

(n = 67) (n = 94) (n = 59) (IBS-C + controls)

C/C 31 28 14 42

C/A 28 49 27 76

A/A 8 17 18 35

P = 0.0019 P = 0.0086

OR = 4.98 OR = 3.23 n.s. Cl = [1.75-14.16 ] Cl = [1.316-7.922]

(IBS-D vs. controls) (IBS-D vs. controls)

[CC] vs [A/A] [CC] vs [A/A]

It is noted that the C/C genotype of the 5-HT3C receptor gene poylmorphism (rs6766410; c.489C>A^*; p.K163N) was also found to be associated with acute chemo- therapy-induced vomiting (CINV) (Fasching PA, Kollmannsberger B, Strissel PL, Ni- esler B, Engel J, Kreis H, Lux MP, Weihbrecht S, Lausen B, Bani MR, Beckmann MW, Strick R. 2008. Polymorphisms in the novel serotonin receptor subunit gene HTR3C show different risks for acute chemotherapy-induced vomiting after anthracycline che- motherapy. J Cancer Res CHn Oncol. 134(10):1079-86).

Example 2 - The HTR3A -42OT (c.-42T) variant causes elevated 5-HT3A receptor density in membranes of transfected HEK293 cells.

In a previous study, we demonstrated that the presence of the -42OT (c.-42T) variant in the 5^'UTR of HTR3A causes higher luciferase reporter gene expression levels compared to the wild-type (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 dis- order. Pharmacogenetics 1 1 :471-475). Here, 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-

3 ing of the 5-HT3 receptor radioligand [ H]GR65630. Maximum binding capacity (Bmax) for cells transfected with the homomeric -42OT receptor was significantly higher (142 ± 17 %, n = 5) than for cells transfected with the homomeric wild-type (c.-42C) receptor (Figure 1 B) whereas radioligand affinity remained unchanged (data not shown). These findings confirm previous results and furthermore implicate that the -42OT variant results in elevated expression levels of 5-HT3A subunits, which can be interpreted as a higher homomeric receptor density on the cell surface.

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.

To investigate putative functional consequences of the HTR3E ^*76G>A (c.^*76A) vari- ant, the wild-type (c.^*76G) and the variant (c.^*76A) full-length 3^'UTR of HTR3E downstream of a luciferase reporter gene were cloned (Figure 3A). Both constructs were transfected into colon carcinoma cells Colo320, but no difference in luciferase activity was detectable (data not shown). By in silico analysis of miRNA binding sites using miRBase, we were able to identify a putative binding domain for hsa-miR-510 (miR- 510; MI0003197) covering bases ^*58 - ^*80 downstream of the stop codon of HTR3E (Figure 2A). The calculated score of 19.12 (19) for putative binding of miR-510 was the highest of 41 predicted miRNAs with calculated scores in the range of 13.94 - 19.12. To confirm a putative HTR3E - miR-510 interaction, the wild-type 3^'UTR luciferase construct was co-transfected with different concentrations of miR-510 precursor mole- cules or same amounts of negative control miRNA with a random sequence (Ambion). Higher concentrations of miR-510 (20 pmol or 50 pmol, but not 2 pmol) led to a significant reduction of luciferase activity to 55 % - 60 % compared to negative control miRNA (100 %; P < 0.001 ) (Figure 2B). These findings confirm the predicted binding of miR-510 to the 3^'UTR of HTR3E and demonstrate a dose-dependent reduction of re- porter gene expression. Since the binding site for miR-510 includes the sequence variant ^*76G>A, it was investigated whether the presence of the ^*76G>A variant interferes with the ability of miR-510 to interact with the 3^'UTR of HTR3E. 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. As predicted, 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.

To further investigate the interaction of miR-510 and HTR3E seen by in vitro analyses, in situ hybridization on human colon tissue sections was performed to check for overlapping expression of these two genes and, in addition, also for HTR3A expression and 5-HT3. All three genes were found to be co-expressed specifically in enterocytes of the colonic mucosa (Figure 4) as well as in myenteric plexuses (not shown). The co- expression of 5-HT3A and 5-HT3E in the respective cells was confirmed by im- munofluorescence experiments using specific antibodies (Figure 4). These results strongly suggest that the expression of the wild-type 5-HT3E subunit is controlled by the co-expressed miR-510 and that 5-HT3 receptors located in the investigated region comprise 5-HT3A-3E subunits.

Example 6 - Screening of the -42C>T variant (c.-42T).

To enable screening for the -42OT variant (c.-42T) in large sample numbers, PCR conditions with the following primers were established:

(i) 59-AGC TGG CCC TTG GTG GGC CCC G-39; and (ii) 59-GCA GAT GGT CAA CCA AGT CC-39.

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. In the presence of the -42C>T variant (c.-42T), 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).

Claims

Claims:

1. Method of diagnosing Irritable Bowel Syndrome in a subject, which comprises (I) obtaining a sample of DNA from the subject; and (II) determining (i) whether said DNA comprises a 5' untranslated region of a 5-

HT3A receptor gene wherein the base at position -42 is a thymine and/or (ii) whether said DNA comprises a 3¹ untranslated region of a 5-HT3E receptor gene wherein the base at position ^*76 is an adenine and/or (iii) whether said DNA comprises a CDS region of a 5-HT3C receptor gene wherein the base at position 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.

2. The method of claim 1 wherein the Irritable Bowel Syndrome is Irritable Bowel Syndrome without constipation.

3. The method of claim 2 wherein the Irritable Bowel Syndrome without constipation is Irritable Bowel Syndrome with diarrhea (IBS-D).

4. The method of any of claims 1 to 3 wherein the subject is a human subject.

5. The method of any of claims 1 to 4, wherein the sample is a body fluid.

6. The method of claim 5, wherein the body fluid is blood.

7. The method of any of claims 1 to 6, wherein DNA is genomic DNA.

8. The method of any of claims 1 to 7, wherein said determination is made by using (i) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to a part of the nucleotide sequence of a 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42 and/or (ii) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical 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/or (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.

9. Use of the method of any one of claims 1 to 8 for diagnosing Irritable Bowel Syn- drome in the subject.

RECTIFIED SHEET (RULE91) ISA/EP

10. 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 (i) whether said DNA comprises a 5' untranslated region of a 5- HT3A receptor gene wherein the base at position -42 is a thymine and/or (ii) whether said DNA comprises a 3' untranslated region of a 5-HT3E receptor gene wherein the base at position *76 is an adenine and/or (iii) whether said DNA comprises a CDS region of a 5-HT3C receptor gene wherein the base at position

489 is a cytosine, the presence of said base(s) indicating the subject's potential benefit.

11. Use of the method of claim 10 for determining whether a subject having or sus- pected of having Irritable Bowel Syndrome may benefit from treatment with a 5-

HT3 antagonist.

12. Nucleic acid comprising the sequence 5'-gtgggcctcgtcctgagcactc-3' (SEQ ID NO:3), or a nucleic acid complementary thereto.

13. Nucleic acid comprising the sequence 5'-cccctttcctaagtaccaacta-3' (SEQ ID NO:4), or a nucleic acid complementary thereto.

14. Use of a nucleic acid comprising (i) a nucleotide sequence essentially comple- mentary or being identical to a part of the nucleotide sequence of a 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42 and/or (ii) a nucleotide sequence essentially complementary or being identical 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/or (iii) 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, for diagnosing Irritable Bowel Syndrome.

5. Use of a nucleic acid comprising (i) a nucleotide sequence essentially comple- mentary or being identical to a part of the nucleotide sequence of a 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42 and/or (ii) a nucleotide sequence essentially complementary or being identical 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/or (iii) a nucleotide sequence essentially complementary or being identical to a part of the nucleotide sequence of a CDS

RECTIFIED SHEET (RULE91) ISA/EP region of a 5-HT3C receptor gene having a cytosine at position 489, for determining whether a subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a 5-HT3 receptor antagonist.

16. The use of claim 14 or 15, wherein the nucleotide sequence is complementary or identical to a nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.

17. Kit comprising (I) (i) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to a part of the nucleotide sequence of a 5' untranslated region of a 5-HT3A receptor gene having a thymine at position -42 and/or (ii) a nucleic acid comprising a nucleotide sequence essentially complementary or being identical to the nucleotide sequence of a 3' untranslated region of a 5-HT3E receptor gene having an adenine at position ^*76 and/or (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, and (II) a means to determine whether the nucleic acid binds to a sample of DNA from a subject.

18. Use of the kit of claim 17 for diagnosing whether a subject has Irritable Bowel Syndrome or may be at risk of developing Irritable Bowel Syndrome.

19. Use of the kit of claim 17 for determining whether the subject having or suspected of having Irritable Bowel Syndrome may benefit from treatment with a 5-HT3 antagonist.

20. Method of treating Irritable Bowel Syndrome in a subject, which comprises admin- istering an effective amount of a 5-HT3 receptor antagonist to the subject, wherein the subject's DNA comprises (i) a 5' untranslated region of a 5-HT3A receptor gene wherein the base at position -42 is a thymine and/or (ii) a 3' untranslated region of a 5-HT3E receptor gene wherein the base at position ^*76 is an adenine and/or (iii) a CDS region of a 5-HT3C receptor gene wherein the base at position 489 is a cytosine.

21. The method of claim 20, wherein the antagonist is selected from the group consisting of proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the functional activity of 5-HT3.

RECTIFIED SHEET^' (RULE91) ISA/EP

22. The method of claim 21 , wherein the functional activity of 5-HT3 is decreased by decreasing the expression of the 5-HT3 receptor, by post- translationally modifiy- ing the 5-HT3 receptor, or by directly interacting with the 5-HT3 receptor.

23. The method of claim 20, wherein the antagonist is an antibody.

24. The method of claim 20, wherein the antagonist is an antisense oligonucleotide.

25. The method of claim 20, wherein the antagonist is a miRNA.

26. The method of claim 20, wherein the antagonist is selected from the group consisting of alosetron, azasetron, bemesetron, BRL-46470, cilansetron, clozapine, dolasetron, fabesetron, galdansetron, GR-65630, granisetron, ICS-205-930, in- disetron, itasetron, lerisetron, lurosetron, LY-278,584, MDL-72222, ondansetron, palonosetron, quipazine, ramosetron, renzapride, ricasetron, SDZ 206-830, tro- pisetron, Y-25130, zacopride, zatosetron, and pharmaceutically acceptable salts thereof.

27. Use of a 5-HT3 receptor antagonist in the manufacture of a medicament for treat- ing Irritable Bowel Syndrome in a subject, wherein the subject's DNA comprises

(i) a 5' untranslated region of a 5-HT3A receptor gene wherein the base at position -42 is a thymine and/or (ii) a 3' untranslated region of a 5-HT3E receptor gene wherein the base at position ^*76 is an adenine and/or (iii) a CDS region of a 5-HT3C receptor gene wherein the base at position 489 is a cytosine.

28. A 5-HT3 receptor antagonist for use in a method of treating Irritable Bowel Syndrome in a subject, wherein the subject's DNA comprises (i) a 5' untranslated region of a 5-HT3A receptor gene wherein the base at position -42 is a thymine and/or (ii) a 3' untranslated region of a 5-HT3E receptor gene wherein the base at position *76 is an adenine and/or (iii) a CDS region of a 5-HT3C receptor gene wherein the base at position 489 is a cytosine.

29. Method of diagnosing Irritable Bowel Syndrome in a subject, which comprises (I) obtaining a sample from the subject; and (II) determining whether expression of a 5-HT3A receptor and/or 5-HT3E receptor gene is upregulated in the sample, the upregulation of the expression indicating that the subject has Irritable Bowel Syndrome or may be at risk of developing Irritable Bowel Syndrome.

RECTIFIED SHEET (RULE91) ISA/EP

30. 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 gastrointestinal tissue sample from the subject; and (II) determining whether expression of a 5-HT3A receptor and/or 5-HT3E receptor gene is upregulated in the sample, the upregulation of the expression indicating the subject's potential benefit.

31. Method of claim 29 or 30, wherein the sample is a gastrointestinal tissue sample.

32. Method of treating Irritable Bowel Syndrome in a subject, which comprises administering an effective amount of a 5-HT3 receptor antagonist to the subject, wherein the subject's expression of a 5-HT3A receptor and/or 5-HT3E receptor gene is upregulated.

RECTIFIED SHEET (RULE91) ISA/EP