WO2002020841A2

WO2002020841A2 - 5-hydroxytryptamine receptor gene polymorphisms and response to treatment

Info

Publication number: WO2002020841A2
Application number: PCT/US2001/027066
Authority: WO
Inventors: Kourtney J Davis; Mary Elizabeth Fling; Arlene R Hughes; Penelope Kupsinel Manasco; Michael James Stubbins
Original assignee: Glaxo Group Limited
Priority date: 2000-09-06
Filing date: 2001-08-31
Publication date: 2002-03-14
Also published as: EP1315837A2; WO2002020841A3; JP2004508056A; AU2001288564A1

Abstract

Correlations between polymorphisms in the 5-hydroxytryptamine 3 receptor gene, and/or polymorphisms in the UGT1A4 gene, and a subject's phenotypic response to treatment with 5-hydroxytryptamine ligands are described. Methods of screening subjects to aid in treatment, methods of screening 5HT ligands, and methods of treatment are presented.

Description

5-HYDROXYTRYPTAMINE RECEPTOR GENE POLYMORPHISMS AND RESPONSE TO TREATMENT

Field of the Invention

The present studies relate to polymorphisms in the 5-hydroxytryptamine 3 receptor (5HT3R) gene, polymorphisms in the UDP-glucuronosyltransferase 1 A4 (UGT1 A4) gene, and phenotypes that are associated or correlated therewith. More particularly, the present studies relate to the correlation of such polymorphisms to the response of subjects with gastrointestinal disorders (such as Irritable Bowel Syndrome (IBS)) to pharmaceutical treatment. The present studies further relate to methods of screening compounds for pharmaceutical activity. The present studies also relate to methods of genotyping subjects for predictive purposes.

Background of the Invention

Many gastrointestinal disorders of unknown etiology, including Irritable Bowel Syndrome (IBS), are believed to be multifactorial disorders. In many of these disorders, no biochemical marker has been found and diagnosis is accomplished primarily by observation of clinical symptoms. Unlike single gene Mendelian disorders, complex disorders such as diabetes, migraine and cardiovascular disease tend to be multifactorial and are caused by the interaction of one or more susceptibility genes with environmental factors. To date, no individual susceptibility genes for IBS have been confirmed by either linkage or association studies

Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain and discomfort, and altered bowel habit. IBS may be characterized by altered bowel habit symptoms of either constipation (constipation predominant subtype) or diarrhea (diarrhea predominant subtype), or alternating constipation and diarrhea (alternator subtype). Currently, there is no single pathophysiological or diagnostic marker of IBS. However, various diagnostic criteria for IBS are available, e.g.., Thompson et al., Gastroent. Int., 2:92 (1989); Manning et al., Br. Med. J. 2:653 (1978); Thompson et al., Gut 45:1143 (1999). Antagonism at 5- hydroxytryptamine (5HT, or serotonin) receptors, such as by alosetron hydrochloride, has been shown to be useful in the treatment of diarrhea-predominant irritable bowel syndrome. Alosetron hydrochloride (CAS registry number: CAS- 122852-69- 1 ; see US Patent

No. 5,360,800) is a 5-HT3 receptor antagonist. Both animal and human studies indicate that 5-HT3 receptor blockade has therapeutic value in the treatment of irritable bowel syndrome, particularly in diarrhea-predominant IBS. (The disclosures of all US patents cited herein are incorporated herein by reference in their entirety.) In double-blind, placebo controlled studies, alosetron hydrochloride has been shown to reduce pain and improve bowel function in female IBS patients whose predominant bowel symptom is diarrhea. See Bardhan et al., Aliment Pharmacol Ther 2000 Jan;14(l):23-34; Jones et al., Aliment Pharmacol Ther 1999 Nov;13(ll):1419-27; Camilleri et al, Aliment Pharmacol Ther 1999 Sep;13(9):l 149-59; Mangel et al., Aliment Pharmacol Ther 1999 May;l3 Suppl 2:77-82. Alosetron has further been suggested as a potential treatment for the symptomatic relief of carcinoid diarrhea. Saslow et al., Gut 1998 May;42(5):628-34.

5-hvdroxytryptamine receptors Molecular cloning has revealed the presence of at least fifteen 5- hydroxytryptamine (5-HT) receptor subtypes, which may be divided into various subfamilies based on structure and function. The 5-hydroxytryptamine receptors are not limited to a single tissue type, but are found in the brain, spinal cord, heart and gastrointestinal tract, as well as other cell types. The 5-HT3 receptors are ligand-gated ion channels (Davies et al., Nature 397:359 (1999)); other 5-HT receptors belong to the superfamily of G-protein-coupled receptors. Gerhardt et al., Eur J Pharmacol 1997 Sep 3;334(l):l-23. Molecular cloning techniques have additionally revealed putative 5-HT receptors for which little or no pharmacological or functional data exist. Sleight et al., Ann N YAcad Sci 1998 Dec 15;861:91-6. In the gastrointestinal tract, various 5-hydroxytryptamine receptors have been identified and characterized, including 5HT3, 5HT4, and 5HTla receptors; these receptors modulate gut motility and visceral sensory pathways. Various 5HT3 receptor antagonists (e.g., alosetron, granisetron and ondansetron) have been identified for the treatment of IBS. This class of drug appears to reduce visceral sensitivity and has inhibitory effects on motor activity in the distal intestine. Full and partial 5HT4 agonists (e.g., HTF919, tegaserod) are potential therapeutics to improve constipation-predominant IBS. Preliminary studies suggest that these agents may have therapeutic potential in IBS. Farthing et al., Baillieres Best Pract Res Clin Gastroenterol. 1999 Oct;13(3):461-71. 5HT4 antagonists (piboserod, SB-207266A) have also been suggested for the treatment of IBS.

UDP-glucuronosyltransferases

The UDP-glucuronosyltransferases (UGTs or UDPGTs; EC 2.4.1.17) catalyze by glucuronidation various endogenous agents, including steroids, bile acids, and bilirubin. The products are rendered more polar, thereby facilitating excretion from the cell. A large family of UGTs exists, allowing for glucuronidation of numerous structurally diverse compounds (Tukey and Strassburg, Ann Rev Pharmacol Toxicol 40:581, 2000). Based upon overall structural similarities, the known UGTs are derived from either the UGT1 or UGT2 gene. The human UGT1 proteins are encoded from a single complex locus on chromosome 2 which contains at least 12 promoters/first exons; each encodes approximately 285 amino acids of the amino-terminal portion of the protein (Ritter et al., J. Biol. Chem 267:3257 (1992); Strassburg et al., Mol. Pharmacol 52:212 (1997)). Exons 2-5 encode the carboxyl terminal half of the UGT1 proteins. The unique UGT1- transcripts are produced by splicing, providing mRNAs that encode different amino- terminal regions but having identical carboxyl-terminal portions. Nomenclature of the UGT gene superfamily is discussed in Mackenzie et al., Pharmacogenetics 7:255 (1997).

Summary of the Invention

The present inventors have determined that polymorphisms in the 5- hydroxytryptamine-3 receptor (5HT3R) gene is correlated with the response of subjects with Irritable Bowel Syndrome (IBS) to pharmaceutical therapy. More particularly, they have found that the C178T polymorphism (as defined herein) in the 5HT3R gene is associated with the response of patients with IBS to treatment with a 5HT antagonist; and have identified a genetic subset of IBS patients that displays a higher incidence of adequate relief of IBS symptoms when treated with alosetron (compared to patients with an alternative polymorphism at the same site of the 5HT3R gene).

A first aspect of the present invention is a method of screening a patient population to identify those subjects with an increased likelihood of responding favorably to treatment with a 5HT ligand for a gastrointestinal disorder. The subjects may have been previously diagnosed as having IBS, or the screening may be used in conjunction with IBS diagnostic efforts.

A further aspect of the present invention is a method of screening a subject suffering from a gastrointestinal disease that is treatable with a 5-hydroxytryptamine (5HT) ligand, as an aid in predicting the subject's response to treatment with a 5HT ligand. The method comprises obtaining a sample of the subject's DNA and determining the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at that site have been associated with different rates of a phenotypic response to treatment with a 5HT ligand. The genotype that is detected in the sample indicates that the subject is likely to have the phenotypic response associated with that genotype. Additionally, the genotype of the subject may be determined at a polymorphic site in the UGT1 A4 gene, where different genotypes at that site have been associated with different rates of phenotypic response to treatment with a 5HT ligand. A further aspect of the present invention is a method of screening a subject with irritable bowel syndrome (IBS), as an aid in predicting the subject's response to treatment with a 5HT ligand. The method comprises obtaining a sample of the subject's DNA and determining the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at that site have been associated with different rates of a phenotypic response to treatment with a 5HT ligand.

A further aspect of the present invention is a method of screening a 5- hydroxytryptamine (5HT) ligand for variations in a measurable phenotypic effects among genetic subpopulations of subjects with a gastrointestinal disorder. The method comprises administering the 5HT ligand to a population of subjects suffering from the gastrointestinal disorder, and obtaining DNA samples from each of the subjects The DNA samples are genotyped for a polymorphic allele of the 5HT3R gene, and correlations between the polymorphic allele genotype and the occurrence of a phenotypic response in the population of subjects are determined. Detection of a genotype that is correlated with an increased or decreased incidence of a desired therapeutic response or a side effect (compared to the incidence in subjects with alternative genotypes) indicates that the effectiveness of the ligand in treating that gastrointestinal disorder varies among genetic subpopulations.

A further aspect of the present invention is a method of treating subjects with Irritable Bowel Syndrome by administering a 5HT3 receptor antagonist, where the patients have a polymorphism in the 5HT3R gene that is predictive of a higher rate of adequate relief of IBS symptoms or a lower incidence of side effects when treated with a 5HT3 receptor antagonist. The rate of relief is increased (and of side effects decreased) compared to subjects who have an alternative polymorphism at the same site of the 5HT3R gene.

A further aspect of the present invention is a method of treating subjects with Irritable Bowel Syndrome by administering alosetron to the subjects, where they have a polymorphism in the 5HT3R gene that is predictive of a higher rate of relief of IBS symptoms and/or a lower incidence of side effects when treated with alosetron (compared to subjects with an alternative polymorphism at the same site of the 5HT3R gene).

A further aspect of the present invention is a method of treating a subject suffering from Irritable Bowel Syndrome (IBS), by identifying the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at this site are associated with different rates of phenotypic response to treatment of IBS with a 5HT receptor ligand. The subject is administered a 5HT receptor ligand that is associated with an increased rate of a favorable phenotypic response in subjects with the identified genotype.

Detailed Description of the Invention

The present invention is concerned with the treatment of gastrointestinal disorders mediated by 5HT receptors, more particularly with the treatment of Irritable Bowel Syndrome (IBS). The present inventors have determined that polymorphic variations in the 5HT3R and UGT1 A4 genes can be correlated to, or associated with, the response to pharmaceutical treatment, particularly treatment with 5HT receptor ligands and more particularly treatment with 5HT3 receptor antagonists including alosetron. The present inventors have identified that polymorphisms in the 5HT3R and UGT1A4 genes are correlated with the response of subjects with a gastrointestinal disorder to treatment with a 5HT receptor ligand. In the present studies, genetic samples were obtained from 237 subjects enrolled in two clinical trials of alosetron for the treatment of non-constipation predominant IBS. The genetic samples were screened for the presence of the C178T polymorphism (as defined herein) in the 5HT3 Receptor (5HT3R) gene, and for the presence of the C70A polymorphism (as defined herein) in the UGT1 A4 gene (as described below), using polymerase chain reaction (PCR) technology as is known in the art.

5HT3R

A nucleotide sequence for the human 5HT3 receptor, as well as exon-intron organization, is disclosed at GenBank accession number AJ005205 (Brass et al., Neuropharmacology 39:308 (2000)). An mRNA sequence for the human 5HT3 receptor is provided at GenBank accession number D49394 (Miyake et al., Mol. Pharmacology

48:407 (1995)), as:

ggaaacatga tccagctgaa ggactgattg caggaaaact tggcagctcc ccaaccttgg -160 tggcccaggg agtgtgaggc tgcagcctca gaaggtgtga gcagtggcca cgagaggcag -100 gctggctggg acatgaggtt ggcagagggc aggcaagctg gcccttggtg ggcctcgccc -40 tgagcactcg gaggcactcc tatgcttgga aagctcgct atg ctg ctg tgg gtc 15

Met Leu Leu Trp Val 1 5 cag cag gcg ctg etc gcc ttg etc etc ccc aca etc ctg gca cag gga 63 Gin Gin Ala Leu Leu Ala Leu Leu Leu Pro T r Leu Leu Ala Gin Gly

10 15 20 gaa gcc agg agg age cga aac ace ace agg ccc get ctg ctg agg ctg 111 Glu Ala Arg Arg Ser Arg Asn Thr Thr Arg Pro Ala Leu Leu Arg Leu 25 30 35 teg gat tac ctt ttg ace aac tac agg aag ggt gtg cgc ccc gtg agg 159 Ser Asp Tyr Leu Leu Thr Asn Tyr Arg Lys Gly Val Arg Pro Val Arg 40 45 50 gac tgg agg aag cca ace ace gta tec att gac gtc att gtc tat gcc 207 Asp Trp Arg Lys Pro Thr Thr Val Ser lie Asp Val lie Val Tyr Ala 55 60 65 ate etc aac gtg gat gag aag aat cag gtg ctg ace ace tac ate tgg 255 lie Leu Asn Val Asp Glu Lys Asn Gin Val Leu Thr Thr Tyr lie Trp 70 75 80 85 tac egg cag tac tgg act gat gag ttt etc cag tgg aac cct gag gac 303 Tyr Arg Gin Tyr Trp Thr Asp Glu Phe Leu Gin Trp Asn Pro Glu Asp 90 95 100 ttt gac aac ate ace aag ttg tec ate ccc acg gac age ate tgg gtc 351 Phe Asp Asn lie Thr Lys Leu Ser lie Pro Thr Asp Ser lie Trp Val 105 110 115 ccg gac att etc ate aat gag ttc gtg gat gtg ggg aag tct cca aat 399 Pro Asp lie Leu lie Asn Glu Phe Val Asp Val Gly Lys Ser Pro Asn 120 125 130 ate ccg tac gtg tat att egg cat caa ggc gaa gtt cag aac tac aag 447 lie Pro Tyr Val Tyr lie Arg His Gin Gly Glu Val Gin Asn Tyr Lys 135 140 145 ccc ctt cag gtg gtg act gcc tgt age etc gac ate tac aac ttc ccc 495

Pro Leu Gin Val Val Thr Ala Cys Ser Leu Asp lie Tyr Asn Phe Pro

150 155 160 165 ttc gat gtc cag aac tgc teg ctg ace ttc ace agt tgg ctg cac ace 543

Phe Asp Val Gin Asn Cys Ser Leu Thr Phe Thr Ser Trp Leu His Thr

170 175 180 ate cag gac ate aac ate tct ttg tgg cgc ttg cea gaa aag gtg aaa 591 lie Gin Asp lie Asn lie Ser Leu Trp Arg Leu Pro Glu Lys Val Lys 185 190 195 tec gac agg agt gtc ttc atg aac cag gga gag tgg gag ttg ctg ggg 639 Ser Asp Arg Ser Val Phe Met Asn Gin Gly Glu Trp Glu Leu Leu Gly 200 205 210 gtg ctg ccc tac ttt egg gag ttc age atg gaa age agt aac tac tat 687 Val Leu Pro Tyr Phe Arg Glu Phe Ser Met Glu Ser Ser Asn Tyr Tyr 215 220 225 gca gaa atg aag ttc tat gtg gtc ate cgc egg egg ccc etc ttc tat 735 Ala Glu Met Lys Phe Tyr Val Val lie Arg Arg Arg Pro Leu Phe Tyr 230 235 240 245 gtg gtc age ctg eta ctg ccc age ate ttc etc atg gtc atg gac ate 783 Val Val Ser Leu Leu Leu Pro Ser He Phe Leu Met Val Met Asp He 250 255 260 gtg ggc ttc tac ctg ccc ccc aac agt ggc gag agg gtc tct ttc aag 831 Val Gly Phe Tyr Leu Pro Pro Asn Ser Gly Glu Arg Val Ser Phe Lys 265 270 275 att aca etc etc ctg ggc tac teg gtc ttc ctg ate ate gtt tct gac 879 He Thr Leu Leu Leu Gly Tyr Ser Val Phe Leu He He Val Ser Asp 280 285 290 acg ctg ccg gcc act gcc ate ggc act cct etc att ggt gtc tac ttt 927 Thr Leu Pro Ala Thr Ala He Gly Thr Pro Leu He Gly Val Tyr Phe 295 300 305 gtg gtg tgc atg get ctg ctg gtg ata agt ttg gcc gag ace ate ttc 975 Val Val Cys Met Ala Leu Leu Val He Ser Leu Ala Glu Thr He Phe 310 315 320 325 att gtg egg ctg gtg cac aag caa gac ctg cag cag ccc gtg cct get 1023 He Val Arg Leu Val His Lys Gin Asp Leu Gin Gin Pro Val Pro Ala 330 335 340 tgg ctg cgt cac ctg gtt ctg gag aga ate gcc tgg eta ctt tgc ctg 1071 Trp Leu Arg His Leu Val Leu Glu Arg He Ala Trp Leu Leu Cys Leu 345 350 355 agg gag cag tea act tec cag agg ccc cca gcc ace tec caa gcc ace 1119 Arg Glu Gin Ser Thr Ser Gin Arg Pro Pro Ala Thr Ser Gin Ala Thr 360 365 370 aag act gat gac tgc tea gcc atg gga aac cac tgc age cac atg gga 1167 Lys Thr Asp Asp Cys Ser Ala Met Gly Asn His Cys Ser His Met Gly 375 380 385 gga ccc cag gac ttc gag aag age ccg agg gac aga tgt age cct ccc 1215 Gly Pro Gin Asp Phe Glu Lys Ser Pro Arg Asp Arg Cys Ser Pro Pro 390 395 400 405 cca cca cct egg gag gcc teg ctg gcg gtg tgt ggg ctg ctg cag gag 1263 Pro Pro Pro Arg Glu Ala Ser Leu Ala Val Cys Gly Leu Leu Gin Glu 410 415 420 ctg tec tec ate egg caa ttc ctg gaa aag egg gat gag ate cga gag 1311 Leu Ser Ser He Arg Gin Phe Leu Glu Lys Arg Asp Glu He Arg Glu 425 430 435 gtg gcc cga gac tgg ctg cgc gtg ggc tec gtg ctg gac aag ctg eta 1359 Val Ala Arg Asp Trp Leu Arg Val Gly Ser Val Leu Asp Lys Leu Leu 440 445 450

ttc cac att tac ctg eta gcg gtg ctg gcc tac age ate ace ctg gtt 1407 Phe His He Tyr Leu Leu Ala Val Leu Ala Tyr Ser He Thr Leu Val 455 460 465 atg etc tgg tec ate tgg cag tac get tga gtgggtacag cccagtggag 1457 Met Leu Trp Ser He Trp Gin Tyr Ala 470 475 gagggggtae agtcctggtt aggtggggae agaggatttc tgcttaggcc cetcaggaee 1517 cagggaatgc cagggacatt ttcaagacac agacaaagtc ccgtgccctg tttccaatgc 1577 caattcatct cagcaatcac aagccaaggt ctgaaccctt ccaccaaaaa ctgggtgttc 1637 aaggccctta eacecttgtc ccacecccag cageteacca tggctttaaa acatgetctc 1697 ttagatcagg agaaactcgg gcactcceta agtccactct agttgtggac ttttccccat 1757 tgaccctcac ctgaataagg gactttggaa ttctgcttct ctttcacaac tttgctttta 1817 ggttgaaggc aaaaccaact ctctactaca caggcctgat aactctgtac gaggcttctc 1877 taacccetag tgtetttttt ttctteacet eacttgtggc agettccctg aacacteatc 1937 ccccatcaga tgatgggagt gggaagaata aaatgcagtg aaaccc 1983

(SEQ ID NO:3 and SEQ ID NO:4).

A polymorphism in the 5' region of the human 5HT3R gene affects position -42

(marking "A" of the atg start codon as +1), wherein the cytosine (c) residue at -42 is replaced by a thymine (t). This nucleotide position is shown above in bold underlined type.

As used herein this polymorphism is referred to as the C178T polymorphism. Polymorphic changes in the 5' nucleotide sequence of a gene may affect transcriptional efficiency.

UGT:

The nucleotide sequence of exon 1 of the human UGT1A4 isozyme is provided at GenBank Accession No. M84128 as:

gtcagatgag cttttcaaga taggcgtgat tggtctttcc cagggttggg cccataacga -170 aaggcagtta tacattaatg ggtaataagt aactggagga gggcactttg tcttccaatt -110 acatgctgat ttgctaggtg gctcaatgac aaggtaatta aggcgaagga aacaaatgta -50 gcaggcacag cgtggggtgg acagtcagct gtcggtggct tctgctgag atg gcc aga 9

Met Ala Arg 1 gga etc cag gtt ccc ctg ccg egg ctg gcc aca gga ctg ctg etc etc 57 Gly Leu Gin Val Pro Leu Pro Arg Leu Ala Thr Gly Leu Leu Leu Leu 5 10 15 etc agt gtc cag ccc tgg get gag agt gga aag gtg ttg gtg gtg ccc 105 Leu Ser Val Gin Pro Trp Ala Glu Ser Gly Lys Val Leu Val Val Pro 20 25 30 35 act gat ggc age ccc tgg etc age atg egg gag gcc ttg egg gag etc 153 Thr Asp Gly Ser Pro Trp Leu Ser Met Arg Glu Ala Leu Arg Glu Leu

40 45 50 cat gcc aga ggc cac cag gcg gtg gtc etc ace cca gag gtg aat atg 201 His Ala Arg Gly His Gin Ala Val Val Leu Thr Pro Glu Val Asn Met 55 60 65 cac ate aaa gaa gag aaa ttt ttc ace ctg aca gcc tat get gtt cca 249 His He Lys Glu Glu Lys Phe Phe Thr Leu Thr Ala Tyr Ala Val Pro 70 75 80 tgg ace cag aag gaa ttt gat cgc gtt acg ctg ggc tac act caa ggg 297 Trp Thr Gin Lys Glu Phe Asp Arg Val Thr Leu Gly Tyr Thr Gin Gly 85 90 95 ttc ttt gaa aca gaa cat ctt ctg aag aga tat tct aga agt atg gca 345 Phe Phe Glu Thr Glu His Leu Leu Lys Arg Tyr Ser Arg Ser Met Ala 100 105 110 115 att atg aac aat gta tct ttg gcc ctt cat agg tgt tgt gtg gag eta 393

He Met Asn Asn Val Ser Leu Ala Leu His Arg Cys Cys Val Glu Leu

120 125 130 ctg cat aat gag gcc ctg ate agg cac ctg aat get act tec ttt gat 441

Leu His Asn Glu Ala Leu He Arg His Leu Asn Ala Thr Ser Phe Asp

135 140 145 gtg gtt tta aca gac ccc gtt aac etc tgt ggg gcg gtg ctg get aag 489 Val Val Leu Thr Asp Pro Val Asn Leu Cys Gly Ala Val Leu Ala Lys 150 155 160 tac ctg teg att cct get gtg ttt ttt tgg agg tac att cca tgt gac 537 Tyr Leu Ser He Pro Ala Val Phe Phe Trp Arg Tyr He Pro Cys Asp 165 170 175 tta gac ttt aag ggc aca cag tgt cca aat cct tec tec tat att cct 585 Leu Asp Phe Lys Gly Thr Gin Cys Pro Asn Pro Ser Ser Tyr He Pro 180 185 190 195 aag tta eta acg ace aat tea gac cac atg aca ttc ctg caa agg gtc 633 Lys Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu Gin Arg Val 200 205 210 aag aac atg etc tac cct ctg gcc ctg tec tac att tgc cat act ttt 681

Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr He Cys His Thr Phe 215 220 225 tct gcc cct tat gca agt ctt gcc tct gag ctt ttt cag aga gag gtg 729 Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gin Arg Glu Val 230 235 240 tea gtg gtg gat ctt gtc age tat gca tec gtg tgg ctg ttc cga ggg 777 Ser Val Val Asp Leu Val Ser Tyr Ala Ser Val Trp Leu Phe Arg Gly 245 250 255 gac ttt gtg atg gac tac ccc agg ccg ate atg ccc aac atg gtc ttc 825 Asp Phe Val Met Asp Tyr Pro Arg Pro He Met Pro Asn Met Val Phe 260 265 270 275 att ggg ggc ate aac tgt gcc aac ggg aag cca eta tct cag 867

He Gly Gly He Asn Cys Ala Asn Gly Lys Pro Leu Ser Gin 280 285 gtctgtattg gtgecttcat ceaatcaatg tteeaggcaa aacacttttt aaaaaatgta 927 tttacttaca agtgcttcca tatctactta tctttccaaa g 968 (SEQ ID NO:l and SEQ ID NO:2). In some individuals a polymorphism is found wherein the twenty-fourth amino acid (indicated by bold type in the above sequence) encoded by exon 1 of UGT1A4 is threonine rather than proline. This occurs when the first nucleotide in the codon is "A" (codon = ace, threonine) rather than "C" (codon = ccc, proline). This polymorphism can be termed the C70A polymorphism; using "a" of the atg start codon as +1, the C→A change occurs at the 70^th nucleotide.

The present inventors determined that, for the C178T polymorphism in the 5HT3R gene, the homozygous genotypes (either homozygous for C at position -42, or homozygous for T at position -42, using the numbering provided above) are associated with an increased rate of adequate relief of IBS symptoms when treated with a 5HT3 receptor antagonist, and therefore an increased incidence of favorable therapeutic response to treatment with a 5HT3 receptor antagonist (compared to subjects with the heterozygous (1,2) genotype treated with the same 5HT3 receptor antagonist, see Table 1). In the present context of treatment of gastrointestinal disease with a 5HT receptor ligand, the C178T polymorphism may be either a marker polymorphism or a functional polymorphism.

Further, a gene-gene interaction was identified. In subjects homozygous (1,1 or 2,2) at the C178T site in the 5HT3R gene, those subjects that were also homozygous (1,1) for the UGT1A4 polymorphism (as defined herein) were more likely to achieve 5-12 weeks of adequate relief during the study, compared to subjects with the heterozygous 1,2 UGT1 A4 genotype. As no subjects had the homozygous 2,2 genotype for UGT1 A4 in the present study, it was not determined how this group of subjects would have responded, hi the present context of treatment of gastrointestinal disease with a 5HT receptor ligand, the C70A polymorphism may be either a marker polymorphism or a functional polymorphism.

According to the present methods, subjects who suffer from a gastrointestinal disease that is treatable with 5HT ligands can be genetically screened, to aid in predicting their response to such treatment. Screening comprises obtaining a sample of DNA from the subject and analyzing the DNA to determine the genotype (presence/absence of polymorphic alleles) at a predetermined polymorphic site in the 5-hydroxytryptamine 3 receptor (5HT3R) gene, where different genotypes at that site have previously been associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease. The genetic screening may further include determination of the genotype (presence/absence of polymorphic alleles) at a predetermined polymorphic site in the UGT1A4 gene, where different genotypes at that site have previously been associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease. The method may include stratifying subjects according to both their 5HT3R genotype and their UGT1 A4 genotype, where a particular combination of polymorphic alleles in these genes has been determined to be associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease. The presence of a particular predetermined genotype therefore indicates an increased likelihood that the individual subject will exhibit the associated phenotype. The genotype will rarely be absolutely predictive, i.e., where a population with a certain genotype displays a high incidence of a particular phenotype, not every individual with that genotype will display the phenotype. However, it will be apparent to those skilled in the art that genotyping a subject as described herein will be an aid in predicting the response a subject will have to treatment with a 5HT ligand, and thus assist in the treatment decision.

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 (1,1), heterozygous (1,2), and homozygous for allele 2 (2,2).

As used herein, a subject that is "predisposed to" a particular phenotypic response based on genotyping of a polymorphic allele will be more likely to display that phenotype than an individual with a different genotype at that polymorphic allele. Where the phenotypic response is based on a biallelic polymorphism, the response may differ among the three possible genotypes.

As used herein, a gastrointestinal disease 'treatable with 5HT ligands' is one in which the administration of a 5HT ligand (in an appropriate pharmaceutical formulation, and in a therapeutically effective amount) has been shown to reduce or alleviate symptoms, without causing unacceptable side effects. Such therapeutic effectiveness is typically evidenced by Regulatory Authority (eg FDA, EMEA) approval of the pharmaceutical preparation, or by publication of the results of clinical studies in peer- reviewed medical journals. Therapeutically effective amounts of such compounds can be readily determined by those skilled in the art using, e.g., dose-response studies. As used herein, the term '5HT ligand' encompasses antagonists and agonists of 5HT receptors, including partial agonists, and drugs that interact with the 5-hydroxytryptamine transporter (5HTT) (e.g., selective serotonin re-uptake inhibitors, SSRIs). 5HT ligands may bind to any subtype of the 5HT receptor, including 5HT3 and 5HT4 receptors; the ligands may be specific for a particular receptor subtype.

Known 5HT-related compounds include 5HT3 receptor antagonists (e.g., alosetron, ondansetron, granisetron, tropisetron, dolasetron, mirtazapine, itasetron, pancopride, zatosetron, azasetron, cliansetron, YM-144 (Yamanouchi) and RS17017 (Roche)). 5HT4 agonists are also known, including tegaserod, prucalopride, norcisapride and the 4-amino-5-chloro-2-methoxy-N-(l -substituted piperidin-4-yl)benzamide known as Y-34959 (Yoshitomi Pharmaceuticals), and buspirone. The use of 5HT4 agonists to treat constipation-predominant IBS has been proposed. 5HT4 antagonists include piboserod (SmithKline Beecham). Dual 5HT3 and 5HT4 receptor agonists include renzapride (SmithKline Beecham) and E3620 (Eisai). A 5HTla receptor agonist is also known, LY315535 (Eli Lilly). Selective serotonin re-uptake inhibitors include fluoxetine, etc. 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. As used herein, a "favorable" phenotypic response to treatment is a response in which adequate relief of a symptom (e.g., pain, urgency, altered bowel habit) is achieved with an acceptable level of side effects. A particular phenotypic response may be more favorable (e.g., achieve a more extensive reduction of symptoms) than another.

Polymorphisms are variant sequences within the human genome that may or may not have a functional consequence. These variants can be used in all aspects of genetic investigation including the analysis and diagnosis of genetic disease, forensics, evolutionary and population studies. Two types of genetic analyses are typically performed: linkage and association studies.

A linkage study provides genetic map information with no prior knowledge or assumption about the function of a gene. In a linkage study one uses DNA polymorphisms to identify chromosomal regions that are identical between affected relatives with the expectation that allele sharing frequencies will be higher for a marker (polymorphism) whose chromosomal location is close to that of the disease allele. Physical cloning of a linkage region narrows down the DNA sequence that could harbor the candidate disease gene. While linkage analysis locates the disease locus to a specific chromosome or chromosome region, the region of DNA in which to search for the gene is typically large, on the order of several million base pairs. h contrast to linkage, association shows the coexistence of a polymorphism and a phenotype in a population. Association studies are based upon linkage disequilibrium, a phenomenon that occurs between a marker and a phenotype if the marker polymorphism is situated in close proximity to the functional polymorphism. Since the marker and functional polymorphism are in close proximity, it requires many generations of recombination to separate them in a population. Thus they tend to co-exist together on the same chromosome at a higher than expected frequency. A marker is said to be associated with a specific phenotype when its frequency is significantly higher among one phenotype group compared to its frequency in another. In general, the closer a marker is to the functionally polymorphic site, the stronger the association.

Association studies offer the opportunity to finely map linkage regions, map loci refractory to linkage analysis and map unknown predisposition loci. Polymorphisms that are in linkage disequilibrium with each other can be spaced over large regions. Linkage disequilibrium has been reported in regions as small as lkilobase or as large as 500 kilobases. Polymorphisms throughout a gene can be in linkage disequilibrium with each other, such that it is valuable to study the whole genome structure - introns, exons, promoters and transcriptional regulatory regions, and 3' and 5' untranslated regions. A marker that is in linkage disequilibrium with a functional polymorphism can be tested for correlation with a phenotype.

The present inventors have determined that polymorphisms in the 5HT3R and the UGT1A4 genes play a role in the response of subjects to pharmaceutical treatment of IBS, thus the genotyping of these genes (either directly or via the gene's expression product) will be useful in identifying therapeutic compounds with measurable effects that vary among subject genotypes. The effect to be measured will depend on the particular gastrointestinal condition, therapeutic compound, and patient population, as will be apparent to one skilled in the art. The measurable effect may be the relief of, or change in, a pathologic sign or symptom or the occurrence of a side effect related to compound administration. Measurement may be objective or subjective (e.g., by patient self- reporting). The association of a 5HT3R and/or UGT1A4 genotype with a therapeutic response will provide a method of determining the probability that an individual subject will respond in a particular way to treatment with 5HT ligands for gastrointestinal disease, hi genotyping, the characteristic that is typically measured is one that can be influenced by a polymorphism in the gene or its expression product. As used herein, the term polymorphism includes Single Nucleotide Polymorphisms (SNPs), insertion/deletion polymorphisms; microsatellite polymorphisms; and variable number of tandem repeat (VNTR) polymorphisms.

According to the present methods, a compound with 5HT ligand activity may be screened for variation in its effects among genetic subpopulations of subjects with a gastrointestinal disorder. Such methods involve administering the compound to a population of subjects suffering from a 5HT-mediated gastrointestinal disorder, obtaining DNA samples from the subjects (which may be done either prior to or after administration of the compound), genotyping polymorphic allelic sites in the 5HT3R gene and/or the UGT1A4 gene, and correlating the genotype of the subjects with their phenotypic responses (both favorable and unfavorable) to the treatment. A genotype that is correlated with an increased rate of a desired therapeutic response (or a decreased rate of an undesirable side effect), compared to the incidence in subjects with alternative genotypes at the polymorphic allelic site, indicates that the effectiveness of the compound in treating such gastrointestinal disorder varies among genetic subpopulations.

Stated another way, the methods of the present invention may be used to determine the correlation of a known 5HT3R polymorphic allele, and/or a known UGT1 A4 polymorphic allele, with the response of subjects with gastrointestinal disorders (such as IBS) to treatment with a 5HT ligand. Subjects with the disease of interest are stratified according to genotype for the particular polymorphic allele, and their response to a therapeutic agent is assessed (either prospectively or retrospectively) and compared among the genotypes. The response to the therapeutic agent may include either, or both, desired therapeutic responses (e.g., the alleviation of signs or symptoms) and undesirable side effects. In this way, genotypes that are associated with an increased (or decreased) rate of therapeutic efficacy, or an increased (or decreased) incidence of a particular side effect, may be identified. The increase or decrease in response is in comparison to the other genotypes, or to a population as a whole.

Polymorphic alleles are typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein from the subject, using any suitable technique as is known in the art. Such a polynucleotide is typically genomic DNA, or a polynucleotide fragment derived from this genomic polynucleotide, such as in a library made using genomic material from the individual (e.g. a cDNA library). The processing of the polynucleotide or protein before the carrying out of the method of the invention is further discussed below. Typically the presence of the polymorphism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymorphism and determining whether the agent binds to the polynucleotide or protein, where the binding indicates that the polymorphism is present. The binding agent may also bind to flanking nucleotides and amino acids on one or both sides of the polymorphism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side, h one embodiment, the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymorphism position. Although the manner of binding will be different than the binding of a polymorphic polynucleotide or protein, this difference will be detectable (for example this may occur in sequence specific PCR as discussed below).

In the case where the presence of the polymorphism is being determined in a polynucleotide it may he detected in the double stranded form, but is typically detected in the single stranded form.

The binding agent may be a polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30, or more nucleotides. The agent may be a molecule that is structurally similar polynucleotides that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing. The agent may be a protein, typically with a length of at least 10 amino acids, such as at least 20, 30, 50, or 100 or more amino acids.. The agent may be an antibody (including a fragment of such an antibody that is capable of binding the polymorphism).

A polynucleotide agent which is used in the method will generally bind to the polymorphism 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 and flanking region.

In one embodiment of the present methods a 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 or protein of the individual. The binding of the probe to the polynucleotide or protein may be used to immobilize either the probe or the polynucleotide or protein (and thus to separate it from one composition or solution).

In another embodiment of the invention the polynucleotide or protein of the individual is immobilized on a solid support and then contacted with the probe. The presence of the probe immobilized 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, hi the case of detecting a polynucleotide polymorphism the solid support is generally made of nitrocellulose or nylon. In the case of a protein polymorphism the method may be based on an ELIS A 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, 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, and therefore the detection of the ligated product may be used to determine the presence of the polymorphism.

In one embodiment the probe is used in a heteroduplex analysis based system to detect polymorphisms, ha 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 sequence- or allele-specific PCR system). Thus a PCR product will only be produced if the polymorphism is present in the polynucleotide of the individual. Thus the presence of the polymorphism may be determined by the detection of the PCR product. Preferably the region of the primer which is complementary to the polymorphism 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 sequence but will not act as a primer for a PCR reaction.

The method may be a Restriction Fragment Length Polymorphism (RFLP) based system. This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognized by a restriction enzyme. Thus treatment of a polynucleotide that has such a polymorphism will lead to different products being produced compared to the corresponding wild-type 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 may be determined based on the change that the presence of the polymorphism makes to the mobility of the polynucleotide or protein during gel electrophoresis. hi the case of a polynucleotide single-stranded conformation polymorphism (SSCP) analysis may be used. This measures the mobility of the single stranded polynucleotide on a denaturing gel compared to the corresponding wild-type polynucleotide, the detection of a difference in mobility indicating the presence of the polymorphism. Denaturing gradient gel electrophoresis 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 polynucleotide indicating the presence of the polymorphism.

The presence of the polymorphism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the TAQMAN™ PCR detection system, hi brief, this assay uses an allele specific primer comprising the sequence around, and including, the polymorphism. The specific primer is labeled with a fluorescent 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.

In the assay, when the allele comprising the polymorphism 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 the 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 the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye in not released from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymorphism can be determined. In another method of detecting the polymorphism a polynucleotide comprising the polymorphic region is sequenced across the region which contains the polymorphism to determine the presence of the polymorphism. Accordingly, any of the following techniques may be utilized in the present methods for genotyping, as is known in the art.

• General: DNA sequencing, sequencing by hybridization;

• Scanning: PTT (Protein truncation technique), 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;

• Hybridization based: solid phase hybridization (dot blots, MASDA, reverse dot blots, oligonucleotide arrays (chips)); solution phase hybridization (TAQMAN™, Molecular Beacons);

• Extension based: ARMS (Amplification Refractory Mutation System), ALEX (Amplification Refractory Mutation System Linear Extension) SBCE (Single Base Chain Extension)

• Incorporation based: Mini-sequencing, APEX; (Arrayed Primer Extension) • Restriction enzyme based: RFLP (restriction fragment length polymorphism)

• Ligation based: OLA (Oligonucleotide Extension Assay)

• Other: Invader (Third Wave Technologies).

The present invention also provides for a predictive (patient care) test or test kit. Such a test will aid in disease management of gastrointestinal disease based on predetermined associations between genotype and phenotypic response to 5HT ligands in treating gastrointestinal disease. Such a test may take different formats, including:

(a) a molecular test which analyses DNA or RNA for the presence of predetermined polymorphisms. An appropriate test kit may include one or more of the following reagents or instruments: a means to detect the binding of the agent 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 a specific polymorphism or genotype and the likelihood that a subject with a gastrointestinal disease such as IBS will respond favorably to therapy with a 5HT ligand;

(b) a biochemical test which analyses materials derived from the subject's body, including proteins or metabolites, that indicate the presence of a pre-determined polymorphism. An appropriate test kit would comprise a molecule, aptamer, peptide or antibody (including an antibody fragment) that specifically binds to a predetermined polymorphic region (or a specific region flanking the polymorphism), or a binding agent as defined herein. The product may additionally comprise one or more additional reagents or instruments (as are known in the art). The test kit would also include printed or machine-readable instructions setting forth the correlation between the presence of a specific polymorphism or genotype and the likelihood that a subject with IBS will respond favorably to therapy with a 5HT ligand.

The present invention provides a method for screening a subject diagnosed with

IBS or another gastrointestinal disorder treatable by 5HT ligands, to determine the likelihood they will respond in a particular way to treatment with a 5HT ligand, more particularly a 5HT3 receptor antagonist, and more particularly alosetron. The method comprises screening the subject for a polymorphism in the 5HT3R gene, and/or the UGT1A4 gene, that have previously been associated with a high or low rate of a particular desirable therapeutic outcome (compared to the rate in subjects with other genotypes), or associated with a high or low incidence of an undesired side effect (compared to the incidence in subjects with other genotypes). Subjects are mammalian, and preferably humans. Treatment of a subject with a 5HT ligand comprises administration of an effective amount of the pharmaceutical agent to a subject in need thereof. The dose of agent is determined according to methods known and accepted in the pharmaceutical arts, and can be determined by those skilled in the art. A suitable dosage range and plasma concentration for alosetron are provided in the disclosure of US Patent Number 5,360,800, the entire disclosure of which is hereby incorporated herein by reference. "Linkage disequilibrium" refers to refers to the tendency of specific alleles at different genomic locations to occur together more frequently than would be expected by chance. Linkage disequilibrium (LD) may be quantitated by methods as are known in the art (see, e.g., Devlin and Risch, Genomics 29:311 (1995); BS Weir, Genetic Data Analysis II, Sinauer Associates, Sunderland, MD (1996)). For example, alleles at given loci may be defined as in complete equilibrium if the frequency of any particular set of alleles (or haplotype) is the product of their individual population frequencies A commonly used measure of linkage disequilibrium is r:

where

*__ = P_A(1-P_A)_{> B} = p_B(l-p_B), D_A = P_AA -p_A ², D_B

1 _AB = ~n_AB -2p_Ap_B n nr² has an approximate chi square distribution with 1 degree of freedom for biallelic markers. Loci exhibiting an r that corresponds to a significiant chi-squared statistic at the 0.05 level may be considered to be in linkage disequilibrium (BS Weir 1996 Genetic Data Analysis II Sinauer Associates, Sunderland, MD). It will be apparent to one skilled in the art that the methods of the present invention may be carried out be detection of an allele that is in linkage disequilibrium, preferably complete linkage disequilibrium, with an allele that has been shown to be associated with a particular phenotype.

The present studies, discussed below, indicate that two candidate genes play a role in the response (adequate relief from pain and discomfort) to treatment with alosetron for Irritable Bowel Syndrome (IBS). Among subjects treated with alosetron, those with the 1,1 or 2,2 5HT3R genotype had a higher rate of response than those with the 1,2 5HT3R genotype. These data do not explain why the heterozygous group had a decreased rate of response.

Among subjects treated with alosetron, results from decision tree analysis and Cbchran-Mantel-Hanzel statistics suggested a gene-gene interaction (5HT3R and UGT1A4), though this was not confirmed by traditional regression modelling. This pattern suggested subjects with 1,1 or 2,2 genotype for 5HT3R-C178T and the 1,1 genotype for UGT1 A4-C70A experienced an increased rate of response, compared with the other genotypes, to treatment with alosetron for IBS.

Among placebo-treated subjects, those with the 1,1 UGTA14-C701A genotype had an increased rate of a more favorable response, compared to subjects with the 1,2 UGTA14 genotype. This gene may be a marker for more moderate disease or for lower pain, as the same 1,1 pattern was associated with an increased rate of response (also seen as part of the gene-gene interaction in the treated group).

EXAMPLES

Example 1: Clinical study and Genotyping

Using genotyping methods as are well known in the art, genetic data were obtained from 237 subjects enrolled in clinical trials of alosetron for the treatment of non- constipation predominant IBS. Samples of subjects' DNA were genotyped, including typing for the presence of the C178T polymorphism in the 5HT3R gene, and for the - presence of the C70A polymorphism in the UGT1 A4 gene (as described above).

In the double-blind, placebo controlled clinical trials, female subjects received up to 12 weeks of treatment with either alosetron or a placebo. A favorable response to alosetron was defined as when a subject reported adequate relief of her IBS symptoms of pain and discomfort; the duration of symptom relief was recorded. Each subject's response to alosetron in the clinical trial setting was then analysed in conjunction with genotyping data, to identify genotypes associated with an increased (or decreased) rate of therapeutic efficacy.

Example 2: Analysis of Clinical Trial Data

The clinical trial data were analyzed, using as the outcome variable 'adequate relief from pain and discomfort' (referred to herein as 'adequate relief). Analyses were conducted using three different coding schemes: continuous (ranging from 0 weeks of adequate relief to 12 weeks of adequate relief, 3 levels (relief occurring for 0 weeks, for 1-4 weeks, or for 5-12 weeks and dichotomous (0-4 weeks of adequate relief, versus 5-12 weeks of adequate relief).

Recursive partitioning analyses (decision trees) were conducted to identify candidate genes associated with treatment response. Two software applications were used for recursive partitioning to compare results. Confirmatory logistic regression, linear regression, and Cochran-Mantel-Hanzel statistics were generated using SAS (Statistical Analysis Software)

Example 3; Recursive Partitioning Results for 3-Level Coding Scheme

Treatment with alosetron (compared to placebo) was the factor most strongly identified with treatment response (p=0.0055).

The recursive partitioning analyses (see Tables 1 & 2) identified polymorphisms in two genes that were associated with an increased rate of response to alosetron in the clinical trials. These were the C178T polymorphism in the 5HT3R gene (as defined herem , and the C70A polymorphism in the UGT1 A4 gene (as defined). Subjects' DNA was coded according to the presence of various polymorphisms, including polymorphisms in the 5HT3R and UGT1A4 genes. A subject homozygous for "C" at the C178T polymorphic site in the 5HT3R gene was coded 1,1; a subject homozygous for "T" at the C178T polymorphic site was coded 2,2; and the heterozygous condition was coded 1,2. A subject homozygous for "C" at the C70A polymorphic site in the UGT1A4 gene was coded 1,1; a subject homozygous for "A" at the C70A polymorphic site was coded 2,2; and the heterozygous condition was coded 1,2.

Where the results for the UGT1 A4 and 5HT3R polymorphisms were missing from a subject's DNA data (coded '0,0'), the data were excluded from the analysis.

For each treatment group, prediction of response by candidate genes was evaluated. (Table 1). Among subjects treated with alosetron,

^■ those heterozygous (1,2) for 5HT3R-C178T were less likely to have a good treatment response compared to subjects homozygous (1,1 or 2,2) at this site.

• Among subjects 1,1 or 2,2 for 5HT3R-C178T, subjects who also were 1,1 for UGTl A4-C70A were more likely to have a good treatment response compared to those with 1,2 for UGTl A4-C70A (p=0.002).

Among subjects treated with placebo,

^■ those with 1,1 for UGTl A4-C70A had a better treatment response than those with 1 ,2 for UGTl A4-C70A (p=0.06)

Table 1: Proportion of subjects with adequate relief (0, 1-4, 5-12 weeks) by treatment and gene status (n=237)

**Results from recursive partitioning

**Results from recursive partitioning

Example 4: Logistic regression results

Logistic regression analyses were used to verify recursive partitioning, with mixed results. Initial logistic models used the three-level outcome response (0, 1-4, 5-12 weeks). The full model including all possible predictor values suggested the only significant predictor of response was the interaction between treatment (alosetron vs. placebo) and the 5HT3R-C178T genotype (p=0.004). Because of this significant interaction term, subsequent analyses were run stratified by treatment group (Table 3).

The model for placebo subjects only showed that neither gene, nor the gene-gene interaction was significant at the p<0.05 level. Borderline in the recursive partitioning analysis, UGTl A4-C70A dropped to p=0.07 in the logistic regression analysis (Table 3). However, note that the trichotomous logistic regression used herein compares the odds of outcome=2 (5-12 weeks) versus 0 (0 weeks) or 1 (1-4 weeks) and the odds of 1 (1-4 weeks) or 2 (5-12 weeks) versus 0 (0 weeks), which is somewhat different from the algorithm used in the decision tree analysis. A change of from p=0.06 to p=0.07, however, suggests a trend of better response among subjects with 1,1 UGT1A4 genotype.

The model for alosetron treated subjects only suggested the same gene-gene interaction as the decision tree, where subjects having a 1,1 UGT1A4 genotype and a

5HT3R genotype of either 1,1 or 2,2, were more likely to have good response when compared to other genotypes (p=0.057). Logistic regression using the dichotomous (binary) endpoint (0-4 weeks versus 5- 12 weeks of adequate relief) revealed no significant genotype effects for the placebo group.

Logistic regression using the dichotomous endpoint (0-4 weeks versus 5-12 weeks of adequate relief) in the alosetron treated group indicated that 5HT3R-C178T was significantly associated with treatment response (p=0.034), but the gene-gene interaction of 5HT3R and UGT1A4 was not significant (Table 3). Cochran-Mantel-Haenszel statistics of response by gene associations, stratified by treatment group, confirmed that 5HT3R and UGTl A4 were significantly associated with response for treated and placebo groups, respectively (Table 5).

Example 5: Linear regression results

Because of the interaction between treatment and candidate genes as described above, models were created separately for subjects on alosetron and placebo, to facilitate interpretation of the overall results.

Among the placebo group, a linear model with the total number of weeks as the endpoint confirmed that subjects with UGT1A4-C70A 1,1 genotype had a better response (higher number of weeks of adequate relief) compared with subjects with 1,2 genotype (p=0.037). Table 4 Among the alosetron treated group the linear model results agreed with the logistic regression and recursive partitioning that subjects with the 5HT3R genotypes 1,1 or 2,2 showed better response (higher number of weeks of adequate relief) than subjects with the 5HT3R 1,2 group (p=0.038), though the gene-gene interaction could not be evaluated due to small numbers. Table 4

adequate relief.

**0-4 versus 5-12 weeks of adequate relief

*** Wald statistic

*t-test

*Fisher's exact test (2-tail) conducted due to small cell sizes EXAMPLE 6: Genotyping of Individuals for 5HT3R and UGT1A4 polymorphisms

DNA samples are obtained from a population of subjects with gastrointestinal disease, and genomic DNA is extracted using standard procedures (automated extraction or using kit formats). The genotypes of the subjects, and any control individuals utilized, are determined for polymorphisms within the 5HT3R and UGT1A4 gene sequences, using either PCR, PCR-RFLP, TAQMAN™ allelic discrimination assays, or any other suitable technique as is known in the art.

If a specific polymorphism resides in an amplification product that is of sufficient physical size (e.g., an insertion/deletion polymorphism of multiple bases), a simple size discrimination assay can be employed to determine the genotype of an individual, hi this case, two primers are employed to specifically amplify the gene of interest in a region surrounding the site of the polymorphism. PCR amplification is carried out, generating products that differ in length, dependent on the genotype (insertion or deletion) they possess. When subjected to gel electrophoresis, the differently sized products are separated, visualized, and the specific genotypes interpreted directly.

PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) assays may also be utilized as is known in the art to detect polymorphisms. For each polymorphic site, a PCR-RFLP assay employs two gene- specific primers to anneal to, and specifically amplify a segment of genomic DNA surrounding the polymorphic site of interest. Following PCR amplification, specific restriction endonuclease enzymes are employed to digest the PCR products produced. The enzyme utilized for an assay is selected due to its specific recognition sequence which it requires to bind to, and cleave the PCR product in the presence/absence of the polymorphism, yielding fragments diagnostic of the specific base present at the polymorphic site. Following cleavage by the restriction enzyme, gel electrophoresis is employed to separate and visualize the fragments produced.

TAQMAN™ (PE Applied Biosystems, Foster City, CA) assays, as are known in the art, may also be utilized to identify polymorphisms. For each polymorphic site the allelic discrimination assay uses two allele specific probes labeled with a different fluorescent dye at their 5' ends but with a common quenching agent at their 3' ends. Both probes have a 3' phosphate group so that Taq polymerase cannot add nucleotides to them. The allele specific probes comprise the sequence encompassing the polymorphic site and differ in the sequence at this site. The allele specific probes are capable of hybridizing without mismatches to the appropriate site.

The allele specific probes are used in conjunction with two primers, one of which hybridizes to the template 5' of the two specific probes, while the other hybridizes to the template 3' of the two probes. If the allele corresponding to one of the specific probes is present, the specific probe will hybridize perfectly to the template. The Taq polymerase, extending the 5' primer, will then remove the nucleotides from the specific probe, releasing both the fluorescent dye and the quenching agent. This will result in an increase in the fluorescence from the dye no longer in close proximity to the quenching agent.

If the allele specific probe hybridizes to the other allele the mismatch at the polymorphic site will inhibit the 5' to 3' endonuclease activity of Taq and hence prevent release of the fluorescent dye.

The ABI7700 sequence detection system is used to measure the increase in the fluorescence from each specific dye at the end of the thermal cycling PCR directly in PCR reaction tubes. The information from the reactions is then analyzed. If an individual is homozygous for a particular allele only fluorescence corresponding to the dye from that specific probe will be released, but if the individual is heterozygous, then both dyes will fluoresce. The genotypes of the individuals are then correlated with their phenotypic response to treatment with a 5HT ligand. Responses that vary among the genetic subpopulations are identified.

Claims

That which is claimed is:

1. A method of screening a subject suffering from a gastrointestinal disease that is treatable with a 5-hydroxytryptamine (5HT) ligand, as an aid in predicting the subject's response to said treatment, comprising:

(a) obtaining a sample of DNA from the subject; and

(b) determining the genotype of said DNA at a polymorphic allelic site in the 5hydroxytryptamine (5HT) receptor gene, where different genotypes at said site are associated with different rates of a phenotypic response to said treatment; where the detected genotype indicates that the subject is likely to have the phenotypic response associated with said genotype.

2. A method according to claim 1, further comprising determining the genotype of said DNA at a polymorphic allelic site in the UGTl A4 gene, where different alleles at said site are associated with different rates of a phenotypic response to said treatment.

3. A method according to claim 2, where said polymorphic allelic site is a C70A polymorphism in the UGTl A4 gene.

4. A method according to claim 1 where said DNA sample is genomic DNA.

5. A method according to claim 1 where said DNA sample is cDNA.

6. A method according to claim 1 where said subject suffers from Irritable Bowel Syndrome (IBS).

7. A method according to claim 1 where said 5HT receptor ligand is a 5HT3 receptor antagonist.

8. A method according to claim 1 where said 5HT receptor ligand is a 5HT4 receptor agonist.

9. A method according to claim 1 where said polymorphic allele is a C178T polymorphism in the 5HT3R gene.

10. A method according to claim 9 where said polymorphic alleles provide different 5' sequences for the 5HT3R gene.

11. A method according to claim 1, further comprising treating said subject with a 5HT receptor ligand.

12. A method according to claim 11 wherein said 5HT receptor ligand is selected from 5HT3 receptor antagonists, 5HT4 antagonists and 5HT4 receptor agonists.

13. A method according to claim 11 where said 5HT receptor ligand is selected from alosetron, ondansetron, and granisetron.

14. A method of screening a subject suffering from irritable bowel syndrome (IBS), as an aid in predicting their response to treatment with a 5-hydroxytryptamine (5HT) receptor ligand, comprising:

(a) obtaining a sample of DNA from the subject; and

(b) genotyping said DNA sample to determine the genotype at a polymorphic allelic site in the 5hydroxytryptamine 3 receptor (5HT3R) gene, where different genotypes at said site are associated with different rates of a phenotypic response to said treatment; where the genotype indicates that the subject is likely to have the phenotypic response associated with said genotype.

15. A method according to claim 14, further comprising determining the genotype of said DNA at a polymorphic allelic site in the UGTl A4 gene, where different alleles at said site are associated with different rates of a phenotypic response to said treatment.

16. A method according to claim 15, where said polymorphic allelic site is a C70A polymorphism in the UGTl A4 gene.

17. A method according to claim 14 where said 5HT receptor ligand is selected from 5HT3 receptor antagonists, 5HT4 antagonists and 5HT4 receptor agonists.

18. A method according to claim 14 where said 5HT receptor ligand is selected from alosetron, ondansetron, and granisetron.

19. A method according to claim 14 where said polymorphism is a C178T polymorphism in the 5HT3R gene.

20. A method according to claim 14 where said polymorphic alleles provide different 5' sequences for the 5HT3R gene.

21. A method according to claim 14 where the genotype indicates the subject is predisposed to relief of IBS symptoms when treated with a 5HT3 receptor antagonist, compared to subjects with alternative genotypes.

22. A method according to claim 14, further comprising treating said subject with a 5HT receptor ligand.

23. A method according to claim 14 wherein said 5HT receptor ligand is selected from 5HT3 receptor antagonists, 5HT4 antagonists and 5HT4 receptor agonists.

24. A method according to claim 14 where said 5HT receptor ligand is selected from alosetron, ondansetron, and granisetron.

25. A method of screening a 5-hydroxytryptamine (5HT) ligand for phenotypic effects in genetic subpopulations of subjects with a gastrointestinal disorder, comprising:

(a) administering said ligand to a population of subjects suffering from said gastrointestinal disorder;

(b) obtaining DNA samples from each of said subjects and genotyping for a polymorphic allele of the 5-hydroxytryptamine 3 receptor (5HT3R) gene; (c) detecting any correlations between the polymorphic allele genotype and the occurrence of a phenotypic response in said population of subjects, where the detection of a genotype that is correlated with an increased or decreased rate of a desired therapeutic response, compared to the rate in subjects with alternative genotypes, indicates that the effectiveness of said ligand in treating said gastrointestinal disorder varies among the genetic subpopulations of said population.

26. A method according to claim 25, further comprising genotyping for a polymorphic allele of the UGT1A4 gene and detecting any correlations between the polymorphic allelic genotypes of the 5HT3R and UGTl A4 genes and the occurrence of a phenotypic response in said population of subject.

27. A method according to claim 26, where said polymorphic allelic site is a C70A polymorphism in the UGTl A4 gene.

28. A method according to claim 25 where said 5HT ligand is selected from 5HT3 receptor antagonists, 5HT4 antagonists and 5HT4 receptor agonists.

29. A method according to claim 25 where said gastrointestinal disorder is irritable bowel syndrome.

30. A method according to claim 25 where said polymorphic variation is a C178T polymorphism in the 5HT3R gene.

31. A method of screening a 5-hydroxytryptamine (5HT ) ligand for phenotypic effects in genetic subpopulations of subjects with a gastrointestinal disorder, comprising:

(a) administering said ligand to a population of subjects suffering from said gastrointestinal disorder,

(b) obtaining DNA samples from each of said subjects and genotyping for a polymorphic allele of the 5-hydroxytryptamine 3 receptor (5HT3R) gene; and

(c) detecting any correlation between the polymorphic allele genotype and the occurrence of a phenotypic response in said population of subjects, where the detection of a genotype that is correlated with an increased or decreased rate of a side effect, compared to the rate in subjects with alternative genotypes, indicates that the effectiveness of said ligand in treating said gastrointestinal disorder varies among the genetic subpopulations of said population.

32. A method according to claim 31, further comprising genotyping for a polymorphic allele of the UGT1A4 gene and detecting any correlations between the polymorphic allelic genotypes of the 5HT3R and UGT1A4 genes and the occurrence of a phenotypic response in said population of subject.

33. A method according to claim 32, where said polymorphic allelic site is a C70A polymorphism in the UGTl A4 gene.

34. A method according to claim 31 where said 5HT ligand is selected from 5HT3 receptor antagonists, 5HT4 receptor antagonists and 5HT4 agonists.

35. A method according to claim 31 where said gastrointestinal disorder is irritable bowel syndrome.

36. A method according to claim 31 where said polymorphic variation is a C178T polymorphism in the 5HT3R gene.

37. A method of treating patients with Irritable Bowel Syndrome (IBS), comprising administering alosetron to patients having a genotype that is associated with an increased rate of relief of IBS symptoms or a decreased rate of a side effect.

38. A method according to claim 37 where said genotype comprises a polymorphism in the 5HT3R gene.

39. A method of treating subjects with Irritable Bowel Syndrome (IBS), comprising administering a 5HT3 receptor antagonist to patients having a polymorphism in the 5HT3 receptor gene that is predictive of an increased rate of relief of IBS symptoms or a decreased rate of a side effect when treated with a 5HT3 receptor antagonist, compared to subjects with an alternative polymorphism at the same site of the 5HT3 receptor gene.

40. A method according to claim 39, where said polymorphism is a C178T polymorphism in the 5HT3R gene.

41. A method according to claim 39, where said 5HT3 receptor antagonist is alosetron.

SEQUENCE LISTING

<110> GLAXO GROUP LIMITED

<120> 5-HYDROXYTRYPTAMINE RECEPTOR GENE POLYMORPHISMS AND RESPONSE TO TREATMENT

<130> PU4151 O

<150> US 60/230,378

<151> 2000-09-06

<160> 4

<170> Patentln version 3.0

<210> 1

<211> 1197

<212> DNA

<213> Homo sapiens .

<220>

<221> CDS

<222> (230) .. (1096)

<400> 1 gtcagatgag cttttcaaga taggcgtgat tggtctttce cagggttggg cecataacga 60 aaggeagtta tacattaatg ggtaataagt aactggagga gggcaetttg tcttccaatt 120 acatgetgat ttgctaggtg gctcaatgac aaggtaatta aggcgaagga aacaaatgta 180 geaggcacag cgtggggtgg acagteaget gteggtggct tetgctgag atg gcc aga 238

Met Ala Arg 1 gga etc cag gtt ccc ctg ccg egg ctg gcc aca gga ctg ctg etc etc 286 Gly Leu Gin Val Pro Leu Pro Arg Leu Ala Thr Gly Leu Leu Leu Leu 5 10 15 etc agt gtc cag ccc tgg get gag agt gga aag gtg ttg gtg gtg ccc 334 Leu Ser Val Gin Pro Trp Ala Glu Ser Gly Lys Val Leu Val Val Pro 20 25 30 35 act gat ggc age ccc tgg etc age atg egg gag gcc ttg egg gag etc 382 Thr Asp Gly Ser Pro Trp Leu Ser Met Arg Glu Ala Leu Arg Glu Leu 40 45 . 50 cat gcc aga ggc cac cag gcg gtg gtc etc ace cca gag gtg aat atg 430 His Ala Arg Gly His Gin Ala Val Val Leu Thr Pro Glu Val Asn Met 55 60 65 cac ate aaa gaa gag aaa ttt ttc ace ctg aca gcc tat get gtt cca 478 His He Lys Glu Glu Lys Phe Phe Thr Leu Thr Ala Tyr Ala Val Pro 70 75 80 tgg ace cag aag gaa ttt gat cgc gtt acg ctg ggc tac act caa ggg 526 Trp Thr Gin Lys Glu Phe Asp Arg Val Thr Leu Gly Tyr Thr Gin Gly 85 90 95 ttc ttt gaa aca gaa cat ctt ctg aag aga tat tct aga agt atg gca 574 Phe Phe Glu Thr Glu His Leu Leu Lys Arg Tyr Ser Arg Ser Met Ala 100 105 110 115 att atg aac aat gta tct ttg gcc ctt cat agg tgt tgt gtg gag eta 622 He Met Asn Asn Val Ser Leu Ala Leu His Arg Cys Cys Val Glu Leu 120 125 130 ctg cat aat gag gcc ctg ate agg cac ctg aat get act tec ttt gat 670 Leu His Asn Glu Ala Leu He Arg His Leu Asn Ala Thr Ser Phe Asp 135 140 145 gtg gtt tta aca gac ccc gtt aac etc tgt ggg gcg gtg ctg get aag 718 Val Val Leu Thr Asp Pro Val Asn Leu Cys Gly Ala Val Leu Ala Lys 150 155 160 tac ctg teg att cct get gtg ttt ttt tgg agg tac att cca tgt gac 766 Tyr Leu Ser He Pro Ala Val Phe Phe Trp Arg Tyr He Pro Cys Asp 165 170 175 tta gac ttt aag ggc aca cag tgt cca aat cct tec tec tat att cct 814 Leu Asp Phe Lys Gly Thr Gin Cys Pro Asn Pro Ser Ser Tyr He Pro 180 185 190 195 aag tta eta acg ace aat tea gac cac atg aca ttc ctg caa agg gtc 862 Lys Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu Gin Arg Val 200 205 210 aag aac atg etc tac cct ctg gcc ctg tec tac att tgc cat act ttt 910 Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr He Cys His Thr Phe 215 220 225 tct gcc cct tat gca agt ctt gcc tct gag ctt ttt cag aga gag gtg 958 Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gin Arg Glu Val 230 235 240 tea gtg gtg gat ctt gtc age tat gca tec gtg tgg ctg ttc cga ggg 1006 Ser Val Val Asp Leu Val Ser Tyr Ala Ser Val Trp Leu Phe Arg Gly 245 250 255 gac ttt gtg atg gac tac ccc agg ccg ate atg ccc aac atg gtc ttc 1054 Asp Phe Val Met Asp Tyr Pro Arg Pro He Met Pro Asn Met Val Phe 260 265 270 275 att ggg ggc ate aac tgt gcc aac ggg aag cca eta tct cag 1096

He Gly Gly He Asn Cys Ala Asn Gly Lys Pro Leu Ser Gin 280 285 gtctgtattg gtgecttcat ceaatcaatg tteeaggcaa aacacttttt aaaaaatgta 1156 tttacttaca agtgcttcca tatctactta tctttccaaa g 1197

<210> 2

<211> 289

<212> PRT

<213> Homo sapiens

<400> 2

Met Ala Arg Gly Leu Gin Val Pro Leu Pro Arg Leu Ala Thr Gly Leu 1 5 10 15

Leu Leu Leu Leu Ser Val Gin Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30

Val Val Pro Thr Asp Gly Ser Pro Trp Leu Ser Met Arg Glu Ala Leu 35 40 45

Arg Glu Leu His Ala Arg Gly His Gin Ala Val Val Leu Thr Pro Glu 50 55 60 Val Asn Met His He Lys Glu Glu Lys Phe Phe Thr Leu Thr Ala Tyr 65 70 75 80

Ala Val Pro Trp Thr Gin Lys Glu Phe Asp Arg Val Thr Leu Gly Tyr 85 90 95

Thr Gin Gly Phe Phe Glu Thr Glu His Leu Leu Lys Arg Tyr Ser Arg 100 105 110

Ser Met Ala He Met Asn Asn Val Ser Leu Ala Leu His Arg Cys Cys 115 120 125

Val Glu Leu Leu His Asn Glu Ala Leu He Arg His Leu Asn Ala Thr 130 135 140

Ser Phe Asp Val Val Leu Thr Asp Pro Val Asn Leu Cys Gly Ala Val 145 150 155 160

Leu Ala Lys Tyr Leu Ser He Pro Ala Val Phe Phe Trp Arg Tyr He 165 170 175

Pro Cys Asp Leu Asp Phe Lys Gly Thr Gin Cys Pro Asn Pro Ser Ser 180 185 190

Tyr He Pro Lys Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu 195 200 205

Gin Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr He Cys 210 215 220

His Thr Phe Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gin 225 230 235 240

Arg Glu Val Ser Val Val Asp Leu Val Ser Tyr Ala Ser Val Trp Leu 245 250 255

Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro He Met Pro Asn 260 265 270

Met Val Phe He Gly Gly He Asn Cys Ala Asn Gly Lys Pro Leu Ser 275 280 285 Gin

<210> 3

<211> 2202

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (220) .. (1653)

<400> 3 ggaaacatga tecagctgaa ggaetgattg caggaaaaet tggcagetec eeaaccttgg 60 tggcccaggg agtgtgaggc tgcagectca gaaggtgtga gcagtggcea cgagaggcag 120 gctggetggg acatgaggtt ggcagagggc aggeaagetg gcccttggtg ggectegccc 180 tgagcactcg gaggcactcc tatgcttgga aagctcgct atg ctg ctg tgg gtc 234

Met Leu Leu Trp Val 1 5 cag cag gcg ctg etc gcc ttg etc etc ccc aca etc ctg gca cag gga 282 Gin Gin Ala Leu Leu Ala Leu Leu Leu Pro Thr Leu Leu Ala Gin Gly 10 15 20 gaa gcc agg agg age cga aac ace ace agg ccc get ctg ctg agg ctg 330 Glu Ala Arg Arg Ser Arg Asn Thr Thr Arg Pro Ala Leu Leu Arg Leu 25 30 35 teg gat tac ctt ttg ace aac tac agg aag ggt gtg cgc ccc gtg agg 378 Ser Asp Tyr Leu Leu Thr Asn Tyr Arg Lys Gly Val Arg Pro Val Arg 40 45 50 gac tgg agg aag cca ace ace gta tec att gac gtc att gtc tat gcc 426 Asp Trp Arg Lys Pro Thr Thr Val Ser He Asp Val He Val Tyr Ala 55 60 65 ate etc aac gtg gat gag aag aat cag gtg ctg ace ace tac ate tgg 474 He Leu Asn Val Asp Glu Lys Asn Gin Val Leu Thr Thr Tyr He Trp 70 75 80 85 tac egg cag tac tgg act gat gag ttt etc cag tgg aac cct gag gac 522 Tyr Arg Gin Tyr Trp Thr Asp Glu Phe Leu Gin Trp Asn Pro Glu Asp 90 95 100 ttt gac aac ate ace aag ttg tec ate ccc acg gac age ate tgg gtc 570 Phe Asp Asn He Thr Lys Leu Ser He Pro Thr Asp Ser He Trp Val 105 110 115 ccg gac att etc ate aat gag ttc gtg gat gtg ggg aag tct cca aat 618 Pro Asp He Leu He Asn Glu Phe Val Asp Val Gly Lys Ser Pro Asn 120 125 130 ate ccg tac gtg tat att egg cat caa ggc gaa gtt cag aac tac aag 666 He Pro Tyr Val Tyr He Arg His Gin Gly Glu Val Gin Asn Tyr Lys 135 140 145 ccc ctt cag gtg gtg act gcc tgt age etc gac ate tac aac ttc ccc 714 Pro Leu Gin Val Val Thr Ala Cys Ser Leu Asp He Tyr Asn Phe Pro 150 155 160 165 ttc gat gtc cag aac tgc teg ctg ace ttc ace agt tgg ctg cac ace 762 Phe Asp Val Gin Asn Cys Ser Leu Thr Phe Thr Ser Trp Leu His Thr 170 175 180 ate cag gac ate aac ate tct ttg tgg cgc ttg cca gaa aag gtg aaa 810 He Gin Asp He Asn He Ser Leu Trp Arg Leu Pro Glu Lys Val Lys 185 190 195 tec gac agg agt gtc ttc atg aac cag gga gag tgg gag ttg ctg ggg 858 Ser Asp Arg Ser Val Phe Met Asn Gin Gly Glu Trp Glu Leu Leu Gly 200 205 210 gtg ctg ccc tac ttt egg gag ttc age atg gaa age agt aac tac tat .906 Val Leu Pro Tyr Phe Arg Glu Phe Ser Met Glu Ser Ser Asn Tyr Tyr 215 220 225 gca gaa atg aag ttc tat gtg gtc ate cgc egg egg ccc etc ttc tat 954 Ala Glu Met Lys Phe Tyr Val Val He Arg Arg Arg Pro Leu Phe Tyr 230 235 240 245 gtg gtc age ctg eta ctg ccc age ate ttc etc atg gtc atg gac ate 1002 Val Val Ser Leu Leu Leu Pro Ser He Phe Leu Met Val Met Asp He 250 255 260 gtg ggc ttc tac ctg ccc ccc aac agt ggc gag agg gtc tct ttc aag 1050 Val Gly Phe Tyr Leu Pro Pro Asn Ser Gly Glu Arg Val Ser Phe Lys 265 270 275 att aca etc etc ctg ggc tac teg gtc ttc ctg ate ate gtt tct gac 1098 He Thr Leu Leu Leu Gly Tyr Ser Val Phe Leu He He Val Ser Asp 280 285 290 acg ctg ccg gcc act gcc ate ggc act cct etc att ggt gtc tac ttt 1146 Thr Leu Pro Ala Thr Ala He Gly Thr Pro Leu He Gly Val Tyr Phe 295 300 305 gtg gtg tgc atg get ctg ctg gtg ata agt ttg gcc gag ace ate ttc 1194 Val Val Cys Met Ala Leu Leu Val He Ser Leu Ala Glu Thr He Phe 310 315 320 325 att gtg egg ctg gtg cac aag caa gac ctg cag cag ccc gtg cct get 1242 He Val Arg Leu Val His Lys Gin Asp Leu Gin Gin Pro Val Pro Ala 330 335 340 tgg ctg cgt cac ctg gtt ctg gag aga ate gcc tgg eta ctt tgc ctg 1290 Trp Leu Arg His Leu Val Leu Glu Arg He Ala Trp Leu Leu Cys Leu 345 350 355 ^a9-7 9 9 9 tea act tec cag agg ccc cca gcc ace tec caa gcc ace 1338 Arg Glu Gin Ser Thr Ser Gin Arg Pro Pro Ala Thr Ser Gin Ala Thr 360 365 370 aag act gat gac tgc tea gcc atg gga aac cac tgc age cac atg gga 1386 Lys Thr Asp Asp Cys Ser Ala Met Gly Asn His Cys Ser His Met Gly 375 380 385 gga ccc cag gac ttc gag aag age ccg agg gac aga tgt age cct ccc 1434 Gly Pro Gin Asp Phe Glu Lys Ser Pro Arg Asp Arg Cys Ser Pro Pro 390 395 400 405 cca cca cct egg gag gcc teg ctg gcg gtg tgt ggg ctg ctg cag gag 1482 Pro Pro Pro Arg Glu Ala Ser Leu Ala Val Cys Gly Leu Leu Gin Glu 410 415 420 ctg tec tec ate egg caa ttc ctg gaa aag egg gat gag ate cga gag 1530 Leu Ser Ser He Arg Gin Phe Leu Glu Lys Arg Asp Glu He Arg Glu 425 430 435 gtg gcc cga gac tgg ctg cgc gtg ggc tec gtg ctg gac aag ctg eta 1578 Val Ala Arg Asp Trp Leu Arg Val Gly Ser Val Leu Asp Lys Leu Leu 440 445 450 ttc cac att tac ctg eta gcg gtg ctg gcc tac age ate ace ctg gtt 1626 Phe His He Tyr Leu Leu Ala Val Leu Ala Tyr Ser He Thr Leu Val 455 460 465 atg etc tgg tec ate tgg cag tac get tgagtgggta cagcccagtg 1673

Met Leu Trp Ser He Trp Gin Tyr Ala 470 475 gaggaggggg tacagtcetg gttaggtggg gaeagaggat ttetgcttag geecctcagg 1733 acecagggaa tgecagggac atttteaaga cacagacaaa gtccegtgec etgtttccaa 1793 tgecaattca teteagcaat cacaagccaa ggtctgaaee ettceaecaa aaaetgggtg 1853 ttcaaggccc ttacaccctt gtcccacccc cagcagctca ccatggcttt aaaacatgct 1913 ctcttagatc aggagaaact cgggcactcc ctaagtccac tctagttgtg gacttttccc 1973 eattgaecct cacctgaata agggactttg gaattctgct tctetttcae aactttgctt 2033 ttaggttgaa ggcaaaaeca actctctaet aeacaggcct gataactetg tacgaggett 2093 etetaacccc tagtgtcttt tttttcttea cctcaettgt ggeagettce etgaacactc 2153 atcccccatc agatgatggg agtgggaaga ataaaatgca gtgaaaccc 2202

<210> 4

<211> 478

<212> PRT

<213> Homo sapiens

<400> 4

Met Leu Leu Trp Val Gin Gin Ala Leu Leu Ala Leu Leu Leu Pro Thr 1 5 10 15

Leu Leu Ala Gin Gly Glu Ala Arg Arg Ser Arg Asn Thr Thr Arg Pro 20 25 30

Ala Leu Leu Arg Leu Ser Asp Tyr Leu Leu Thr Asn Tyr Arg Lys Gly 35 40 45

Val Arg Pro Val Arg Asp Trp Arg Lys Pro Thr Thr Val Ser He Asp 50 55 60

Val He Val Tyr Ala He Leu Asn Val Asp Glu Lys Asn Gin Val Leu 65 70 75 80

Thr Thr Tyr He Trp Tyr Arg Gin Tyr Trp Thr Asp Glu Phe Leu Gin 85 90 95

Trp Asn Pro Glu Asp Phe Asp Asn He Thr Lys Leu Ser He Pro Thr 100 105 110

Asp Ser He Trp Val Pro Asp He Leu He Asn Glu Phe Val Asp Val 115 120 125

Gly Lys Ser Pro Asn He Pro Tyr Val Tyr He Arg His Gin Gly Glu 130 135 140 Val Gin Asn Tyr Lys Pro Leu Gin Val Val Thr Ala Cys Ser Leu Asp 145 150 155 160

He Tyr Asn Phe Pro Phe Asp Val Gin Asn Cys Ser Leu Thr Phe Thr 165 170 175

Ser Trp Leu His Thr He Gin Asp He Asn He Ser Leu Trp Arg Leu 180 185 190

Pro Glu Lys Val Lys Ser Asp Arg Ser Val Phe Met Asn Gin Gly Glu 195 200 205

Trp Glu Leu Leu Gly Val Leu Pro Tyr Phe Arg Glu Phe Ser Met Glu 210 215 220

Ser Ser Asn Tyr Tyr Ala Glu Met Lys Phe Tyr Val Val He Arg Arg 225 230 235 240

Arg Pro Leu Phe Tyr Val Val Ser Leu Leu Leu Pro Ser He Phe Leu 245 250 255

Met Val Met Asp He Val Gly Phe Tyr Leu Pro Pro Asn Ser Gly Glu 260 265 270

Arg Val Ser Phe Lys He Thr Leu Leu Leu Gly Tyr Ser Val Phe Leu 275 280 285

He He Val Ser Asp Thr Leu Pro Ala Thr Ala He Gly Thr Pro Leu 290 295 300

He Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu Val He Ser Leu 305 310 315 320

Ala Glu Thr He Phe He Val Arg Leu Val His Lys Gin Asp Leu Gin 325 330 335

Gin Pro Val Pro Ala Trp Leu Arg His Leu Val Leu Glu Arg He Ala 340 345 350

Trp Leu Leu Cys Leu Arg Glu Gin Ser Thr Ser Gin Arg Pro Pro Ala 10

355 360 365

Thr Ser Gin Ala Thr Lys Thr Asp Asp Cys Ser Ala Met Gly Asn His 370 375 380

Cys Ser His Met Gly Gly Pro Gin Asp Phe Glu Lys Ser Pro Arg Asp 385 390 395 400

Arg Cys Ser Pro Pro Pro Pro Pro Arg Glu Ala Ser Leu Ala Val Cys 405 410 415

Gly Leu Leu Gin Glu Leu Ser Ser He Arg Gin Phe Leu Glu Lys Arg 420 425 430

Asp Glu He Arg Glu Val Ala Arg Asp Trp Leu Arg Val Gly Ser Val 435 440 445

Leu Asp Lys Leu Leu Phe His He Tyr Leu Leu Ala Val Leu Ala Tyr 450 455 460

Ser He Thr Leu Val Met Leu Trp Ser He Trp Gin Tyr Ala 465 470 475