GENOTYPING HUMAN UDP-GLUCURONOSYLTRANSFERASE 2B4 (UGT2B4), 2B7 (UGT2B7) AND 2B15 (UGT2B15) GENES

Abstract: Genetic polymorphisms are identified in the human UGT2B4, UGT2B7 and UGT2B15 genes that alter UGT2B activity. Nucleic acids comprising the polymorphic sequences are used to screen patients for altered metabolism for UGT2B substrates, potential drug-drug interactions, and adverse/side effects, as well as diseases that result from environmental or occupational exposure to toxins. The nucleic acids are used to establish animal, cell and in vitro models for drug metabolism.

Description: GENOTYPING HUMAN UDP-GLUCURONOSYLTRANSFERASE 2B4 (UGT2B4), 2B7 (UGT2B7) and 2B15 (UGT2B15) GENES INTRODUCTION The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reactions (phase I reactions), in which lipophilic compounds are modified via monooxygenation, dealkylation, reduction, aromatization, or hydrolysis. These modified molecules can then be substrates for the phase II reactions, often called conjugation reactions, as they conjugate a functional group with a polar, endogenous compound. Drug glucuronidation, a major phase II conjugation reaction in the mammalian detoxification system, is catalyzed by the UDP-glucuronosyltransferases (UGTs) (Batt AM, et al. (1994) Clin Chim Acta 226:171-190; Burchell et al. (1995) Life Sci. 57:1819- 31). The UGTs are a family of enzymes that catalyze the glucuronic acid conjugation of a wide range of endogenous and exogenous substrates including phenols, alcohols, amines and fatty acids. The reactions catalyzed by UGTs permit the conversion of a large range of toxic endogenous/xenobiotic compounds to more water-soluble forms for subsequent excretion (Parkinson A (1996) Toxicol Pathol 24:48-57). The UGT isoenzymes are located primarily in hepatic endoplasmic reticulum and nuclear envelope (Parkinson A (1996) Toxicol Pathol 24:48-57), though they are also expressed in other tissues such as kidney and skin. UGTs are encoded by a large multigene superfamily that has evolved to produce catalysts with differing but overlapping substrate specificities. Three families, UGT1, UGT2, and UGT8, have been identified within the superfamily. UGTs are assigned to one the subfamilies based on amino acid sequence identity, e.g., UGT1 family members have greater than 45% amino acid sequence identity (Mackenzie et al. (1997) Pharmacogenetics 7:255-69). A single gene encodes several human UGT1 isoforms, the substrate specificity of each of which is thought to arise from differential splicing of a number of substrate-specific 5-prime regions of a single mRNA transcript to a shared 3-prime portion. On the other hand, members of the mammalian UGT2 gene subfamily, which encode the odorant and steroid-metabolizing isoforms, show nucleotide differences in sequence throughout the length of the cDNAs. This suggested that the UGT2 isoenzymes are encoded by several independent genes. The UGT2 genes have been further divided on the basis of their tissue-specific expression patterns into the UGT2A gene subfamily, which encodes olfactory-specific isoforms, and the UGT2B gene subfamily, which encodes steroid-metabolizing isoforms in the liver. Monaghan et al. (1994) Genomics 23:496-499 mapped the UGT2B9 and the UGT2B15 genes to chromosome 4q13, giving a provisional ordering of the genes as UGT2B9-UGT2B4-UGT2B15. The UGT2B subfamily contains phenobarbital-inducible genes, as well as numerous genes that are constitutively expressed and are involved in the glucuronidation of endogenous steroids and biogenic amines (Mackenzie, et al. supra.) Evidence suggests that UGT2B4 is exclusively expressed in human liver, and not in human kidney. Levesque et al. (1997) Pharmacogenetics 7:317; and Coffman et al. (1997) Drug Metabol. and Dispos. 25:1-4, describe UGT2B gene polymorphisms. Alteration of the expression or function of UGTs may affect drug metabolism. For example, there may be common polymorphisms in the human UGT2B gene that alter expression or function of the protein product and cause drug exposure-related phenotypes. Thus, there is a need in the field to identify UGT2B polymorphisms in order to provide a better understanding of drug metabolism and the diagnosis of drug exposure-related phenotypes. SUMMARY OF THE INVENTION Genetic sequence polymorphisms are identified in the UGT2B4, UGT2B7 and UGT2B15 genes, herein generically referred to as "UGT2B genes". Nucleic acids comprising the polymorphic sequences are used in screening assays, and for genotyping individuals. The genotyping information is used to predict an individuals' rate of metabolism for UGT2B substrates, potential drug-drug interactions, and adverse/side effects. Specific polynucleotides include the polymorphic UGT2B4 sequences set forth in SEQ ID NOs:25-38; the polymorphic UGT2B7 sequences set forth in SEQ ID NOs:84-111 ; and the polymorphic UGT2B15 sequences set forth in SEQ ID NOs: 147-164. The nucleic acid sequences of the invention may be provided as probes for detection of UGT2B locus polymorphisms, where the probe comprises a polymorphic sequence of SEQ ID NOs:25-38; 84-111 and 147-164. The sequences may further be utilized as an array of oligonucleotides comprising two or more probes for detection of UGT2B locus polymorphisms. Another aspect of the invention provides a method for detecting in an individual a polymorphism in UGT2B metabolism of a substrate, where the method comprises analyzing the genome of the individual for the presence of at least one UGT2B polymorphism; wherein the presence of the predisposing polymorphism is indicative of an alteration in UGT2B expression or activity. The analyzing step of the method may be accomplished by detection of specific binding between the individual's genomic DNA with an array of oligonucleotides comprising UGT2B locus polymorphic sequences. In other embodiments, the alteration in UGT2B expression or activity is tissue specific, or is in response to a UGT2B modifier that induces or inhibits UGT2B expression. BRIEF DESCRIPTION OF THE SEQUENCE LISTING UGT2B Reference Sequences. SEQ ID NOs: 1-6 list the sequence of the reference UGT2B4 exons, where exon 1 is SEQ ID NO:1 , exon 2 is SEQ ID NO:2 and so forth. Partial sequence of the flanking introns is included; the boundaries are annotated in the SEQLIST. The cDNA sequence is set forth in SEQ ID NO:7, and the encoded amino acid sequence in SEQ ID NO:8. SEQ ID NO:39 lists the sequence of the UGT2B7 cDNA sequence, the encoded polypeptide is provided in SEQ ID NO:40. SEQ ID NOs: 41-45 list the sequence of the reference UGT2B7 exons, where exon 1 is SEQ ID NO:41, exon 2 is SEQ ID NO:42 and so forth. Partial sequence of the flanking introns is included; the boundaries are annotated in the SEQLIST. SEQ ID NO:112 lists the sequence of the UGT2B15 cDNA sequence, the encoded polypeptide is provided in SEQ ID NO:113. SEQ ID NOs:114-118 list the sequence of the reference UGT2B15 exons, where exon 1 is SEQ ID NO: 114, exon 2 is SEQ ID NO:115 and so forth. Partial sequence of the flanking introns is included; the boundaries are annotated in the SEQLIST. Primers. The PCR primers for amplification of polymorphic sequences are set forth as SEQ ID NOs:9-14; 46-66; and 135-146. The primers used in sequencing isolated polymorphic sequences are presented as SEQ ID NOs:15-24; 67-83; and 119-134. Polymorphisms. Polymorphic sequences of UGT2B4 are presented as SEQ ID NOs:25-38. Polymorphic sequences of UGT2B7 are presented as SEQ ID NOs:84-111. Polymorphic sequences of UGT2B15 are presented as SEQ ID NO:147-164. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Pharmacogenetics is the association between an individual's genotype and that individual's ability to metabolize or react to a therapeutic agent. Differences in metabolism or target sensitivity can lead to severe toxicity or therapeutic failure by altering the relation between bioactive dose and blood concentration of the drug. Relationships between polymorphisms in metabolic enzymes or drug targets and both response and toxicity can be used to optimize therapeutic dose administration. Genetic polymorphisms are identified in the UGT2B4, UGT2B7 and UGT2B15 genes. Nucleic acids comprising the polymorphic sequences are used to screen patients for altered metabolism for UGT2B substrates, potential drug-drug interactions, and adverse/side effects, as well as diseases that result from environmental or occupational exposure to toxins. The nucleic acids are used to establish animal, cell culture and in vitro cell-free models for drug metabolism. Definitions It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a construct" includes a plurality of such constructs and reference to "the UGT2B nucleic acid" includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise. UGT2B4 reference sequence. The sequence of human UGT2B4 cDNA may be accessed through Genbank, accession number Y00317, and is provided in SEQ ID NOs:1-7. The amino acid sequence of UGT2B4 is listed as SEQ ID NO:8. The sequence of human UGT2B7 may be accessed through Genbank, accession number 600068, and in the SEQLIST as described above. The sequence of human UGT2B15 may be accessed through Genbak, accession number 600069, and in the SEQLIST as described above. The nucleotide sequences provided herein differ from the published sequence at certain positions throughout the sequence. Where there is a discrepancy the provided sequence is used as a reference. The term "wild-type" may be used to refer to the reference coding sequences of UGT2B4, UGT2B7 and UGT2B15, and the term "variant", or "UGT2B" to refer to the provided variations in the UGT2B sequences. The UGT2B4, UGT2B7 and UGT2B15 sequences are generically referred to as "UGT2B", and may be further distinguished by the species, e.g. human, mouse, etc., or by the specific gene number, e.g. UGT2B4, UGT2B7, etc. Where there is no published form, such as in the intron sequences, the term wild-type may be used to refer to the most commonly found allele. It will be understood by one of skill in the art that the designation as "wild-type" is merely a convenient label for a common allele, and should not be construed as conferring any particular property on that form of the sequence. UGT2B polymorphic sequences. It has been found that specific sites in the UGT2B4, UGT2B7 and UGT2B15 genes sequence are polymorphic, i.e. within a population, more than one nucleotide (G, A, T, C) is found at a specific position. Polymorphisms may provide functional differences in the genetic sequence, through changes in the encoded polypeptide, changes in mRNA stability, binding of transcriptional and translation factors to the DNA or RNA, and the like. The polymorphisms are also used as single nucleotide polymorphisms (SNPs) to detect genetic linkage to phenotypic variation in activity and expression of the particular UGT2B protein. SNPs are generally biallelic systems, that is, there are two alleles that an individual may have for any particular marker. SNPs, found approximately every kilobase, offer the potential for generating very high density genetic maps, which will be extremely useful for developing haplotyping systems for genes or regions of interest, and because of the nature of SNPs, they may in fact be the polymorphisms associated with the disease phenotypes under study. The low mutation rate of SNPs also makes them excellent markers for studying complex genetic traits. SNPs are provided in the UGT2B4, UGT2B7 and UGT2B15 intron and exon sequences. Tables 4, 7 and 10, and the corresponding sequence listing, provide both forms of each polymorphic sequence. For example, SEQ ID NO:37 and 38 are the alternative forms of a single polymorphic site. The provided sequences also encompass the complementary sequence corresponding to any of the provided polymorphisms. In order to provide an unambiguous identification of the specific site of a polymorphism, sequences flanking the polymorphic site are shown in the tables, where the 5' and 3' flanking sequence is non-polymorphic, and the central position, shown in bold, is variable. It will be understood that there is no special significance to the length of non- polymorphic flanking sequence that is included, except to aid in positioning the polymorphism in the genomic sequence. The UGT2B exon sequences have been published, and therefore one of each pair of the sequences from exons in Tables 4, 7 and 10 are publically known sequence. The intron sequence has not been published, and hence both forms of this polymorphic sequence is novel. As used herein, the term "UGT2B4, UGT2B7 and UGT2B15 genes" is intended to generically refer to both the wild-type and variant forms of the sequence, unless specifically denoted otherwise. As it is commonly used in the art, the term "gene" is intended to refer to the genomic region encompassing the 5' UTR, exons, introns, and the 3' UTR. Individual segments may be specifically referred to, e.g. exon 2, intron 5, etc. Combinations of such segments that provide for a complete UGT2B protein may be referred to generically as a protein coding sequence. Nucleic acids of interest comprise the provided UGT2B V nucleic acid sequence(s), as set forth in Tables 4, 7 and 10. Such nucleic acids include short hybridization probes, protein coding sequences, variant forms of UGT2B cDNA, segments, e.g. exons, introns, etc., and the like. Methods of producing nucleic acids are well-known in the art, including chemical synthesis, cDNA or genomic cloning, PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 20 nt, often at least 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide, promoter motifs, etc. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. The UGT2B nucleic acid sequences are isolated and obtained in substantial purity, generally as other than an intact or naturally occurring mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a UGT2B sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome. For screening purposes, hybridization probes of the polymorphic sequences may be used where both forms are present, either in separate reactions, spatially separated on a solid phase matrix, or labeled such that they can be distinguished from each other. Assays may utilize nucleic acids that hybridize to one or more of the described polymorphisms. An array may include all or a subset of the polymorphisms listed in Tables 4, 7 and 10. One or both polymorphic forms may be present in the array, for example the polymorphism of SEQ ID NO:37 and 38 may be represented by either, or both, of the listed sequences. Usually such an array will include at least 2 different polymorphic sequences, i.e. polymorphisms located at unique positions within the locus, and may include all of the provided polymorphisms. Arrays of interest may further comprise sequences, including polymorphisms, of other genetic sequences, particularly other sequences of interest for pharmacogenetic screening, e.g. UGT1, other UGT2 sequences, cytochrome oxidase polymorphisms, ete. The oligonucleotide sequence on the array will usually be at least about 12 nt in length, may be the length of the provided polymorphic sequences, or may extend into the flanking regions to generate fragments of 100 to 200 nt in length. For examples of arrays, see Ramsay (1998) Nat. Biotech. 16:40-44; Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics 14:457-460. Nucleic acids may be naturally occurring, e.g. DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O- phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-O- methyl or 2'-O-allyl sugars, which provides resistance to degradation without compromising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'- deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'- deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively. UGT2B polypeptides. A subset of the provided nucleic acid polymorphisms in UGT2B exons confer a change in the corresponding amino acid sequence. Using the amino acid sequence provided in SEQ ID NO:8 as a reference for UGT2B4, the amino acid polymorphisms of the invention include lys→asn, pos. 40; and glu— asp, pos. 454. Using the amino acid sequence provided in SEQ ID NO:40 as a reference for UGT2B7, the amino acid polymorphisms of the invention include leu→phe, pos. 107; thr— ile, pos. 179; and lys-→gln, pos. 430. Using the amino acid sequence provided in SEQ ID NO: 125 as a reference for UGT2B15, the amino acid polymorphisms of the invention include ser—gly, pos. 15; asp→tyr, pos. 85; leu→pro, pos. 170; his→gln, pos. 282; ala→val, pos. 398; val→ile, pos. 443; and thr→lys, pos. 523. Polypeptides comprising at least one of the provided polymorphisms (UGT2B V polypeptides) are of interest. The term "UGT2B V polypeptides" as used herein includes complete UGT2B protein forms, e.g. such splicing variants as known in the art, and fragments thereof, which fragments may comprise short polypeptides, epitopes, functional domains; binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof. Polypeptides will usually be at least about 8 amino acids in length, more usually at least about 12 amino acids in length, and may be 20 amino acids or longer, up to substantially the complete protein. The UGT2B4, UGT2B7 and UGT2B15 genetic sequences, including polymorphisms, may be employed for polypeptide synthesis. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed that are functional in the expression host. The polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. Small peptides can also prpared by chemical synthesis. Substrate. A substrate is a chemical entity that is modified by UGT2B4, UGT2B7 or UGT2B15, usually under normal physiological conditions. Although the duration of drug action tends to be shortened by metabolic transformation, drug metabolism is not "detoxification". Frequently the metabolic product has greater biologic activity than the drug itself. In some cases the desirable pharmacologic actions are entirely attributable to metabolites, the administered drugs themselves being inert. Likewise, the toxic side effects of some drugs may be due in whole or in part to metabolic products. Substrates can be either endogenous substrates, i.e. substrates normally found within the natural environment of UGT2B, such as estriol, or exogenous, i.e. substrates that are not normally found within the natural environment of UGT2B. UGT2B catalyzes glucuronidation of its substrates. The enzymes are specific for UDP-glucuronic acid, and not other UDP sugars. Exemplary UGT2B4 substrates (i.e., substrates of wild-type UGT2B4 and/or UGT2B4 V polypeptides) include, but are not necessarily limited to estriol and the catechol estrogens 4-hydroxyestrone, and 2-hydroxyestriol, 2-aminophenol, 4-methylumbellifereone, 1-naphthol, 4-hydroxybiphenyl and 4-nitrophenol, 2-aminophenol, 4-hydroxybiphenyl, menthol, etc., among other substrates (Burchell et al. (1991) DNA Cell Biol 10:487-494, Jin CJ, et al. (1993) Biochem Biophvs Res Commun 194:496-503). Exemplary UGT2B7 substrates (i.e., substrates of wild-type UGT2B7 and/or UGT2B7 V polypeptides) include, but are not necessarily limited to oxazepam, hyodeoxycholic acid, estriol, S-naproxen, ketoprofen, ibuprofen, fenoprofen, clofibric acid (Patel et a/ (1995) Pharmacogenetics 5(1):43-49), morphine (Coffman et al (1997) Drug Metabolism and Disposition 25:1-4), DMXAA (5,6-dimethylxantheonone-4-acetic acid) (Miners et al (1997) Cancer Res 57:284), 2-Hydroxy AAF, 4 methylumbelliferone, carboxylic acid drugs (BP-7,8- trans diol) (Burchell et al., supra.) Exemplary UGT2B15 substrates (i.e., substrates of wild-type UGT2B15 and/or UGT2B15 V polypeptides) include, but are not necessarily limited to 4-hydroxybiphenyl, 1- naphthol, 4 methylumbelliferone , naringenin, eugenol (Burchell et al., supra.), simple phenolic compounds, 7-hydroxylated coumarins, flavonoids, anthraquinones; endogenous estrogens and androgens (Green et al. (1994) Drug Metabolism and Disposition 22:799. Modifier. A modifier is a chemical agent that modulates the action of a UGT2B molecule, either through altering its enzymatic activity (enzymatic modifier) or through modulation of expression (expression modifier, e.g., by affecting transcription or translation). In some cases the modifier may also be a substrate. Pharmacokinetic parameters. Pharmacokinetic parameters provide fundamental data for designing safe and effective dosage regimens. A drug's volume of distribution, clearance, and the derived parameter, half-life, are particularly important, as they determine the degree of fluctuation between a maximum and minimum plasma concentration during a dosage interval, the magnitude of steady state concentration and the time to reach steady state plasma concentration upon chronic dosing. Parameters derived from in vivo drug administration are useful in determining the clinical effect of a particular UGT2B genotype. Expression assay. An assay to determine the effect of a sequence polymorphism on UGT2B expression. Expression assays may be performed in cell-free extracts, or by transforming cells with a suitable vector. Alterations in expression may occur in the basal level that is expressed in one or more cell types, or in the effect that an expression modifier has on the ability of the gene to be inhibited or induced. Expression levels of a variant alleles are compared by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like. Gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins (Ausubel, F.M. et al. (1989) In: Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, New York). This method has been used widely in the study of sequence-specific DNA-binding proteins, such as transcription factors. The assay is based on the observation that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The gel shift assay is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or cell extract preparations), with an end-labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated DNA sequences. Expression assays can be used to detect differences in expression of polymorphisms with respect to tissue specificity, expression level, or expression in response to exposure to various substrates, and/or timing of expression during development. For example, since UGT2B4 is expressed in liver, polymorphisms could be evaluated for expression in tissues other than liver, or expression in liver tissue relative to a reference UGT2B4 polypeptide. Substrate screening assay. Substrate screening assays are used to determine the metabolic activity of a UGT2B protein or peptide fragment on a substrate. Many suitable assays are known in the art, including the use of primary or cultured cells, genetically modified cells (e.g., where DNA encoding the UGT2B polymorphism to be studied is introduced into the cell within an artificial construct), cell-free systems, e.g. microsomal preparations or recombinantly produced enzymes in a suitable buffer, or in animals, including human clinical trials (see, e.g., Burchell et al. (1995) Life Sci. 57:1819-1831 , specifically incorporated herein by reference. Where genetically modified cells are used, since most cell lines do not express UGT2B activity (liver cells lines being the exception), introduction of artificial construct for expression of the UGT2B polymorphism into many human and non- human cell lines does not require additional modification of the host to inactivate endogenous UGT2B expression/activity. Clinical trials may monitor serum, urine, etc. levels of the substrate or its metabolite(s). Typically a candidate substrate is input into the assay system, and the conversion to a metabolite is measured over time. The choice of detection system is determined by the substrate and the specific assay parameters. Assays are conventionally run, and will include negative and positive controls, varying concentrations of substrate and enzyme, etc. Genotyping: UGT2B genotyping is performed by DNA or RNA sequence and/or hybridization analysis of any convenient sample from a patient, e.g. biopsy material, blood sample (serum, plasma, etc.), buccal cell sample, etc. A nucleic acid sample from an individual is analyzed for the presence of polymorphisms in UGT2B, particularly those that affect the activity or expression of UGT2B. Specific sequences of interest include any polymorphism that leads to changes in basal expression in one or more tissues, to changes in the modulation of UGT2B expression by modifiers, or alterations in UGT2B substrate specificity and/or activity. Linkage Analysis: Diagnostic screening may be performed for polymorphisms that are genetically linked to a phenotypic variant in UGT2B activity or expression, particularly through the use of microsatellite markers or SNPs. The microsatellite marker or SNP itself may not phenotypically expressed, but is linked to sequences that result in altered activity or expression. Two polymorphic variants may be in linkage disequilibrium, i.e. where alleles show non-random associations between genes even though individual loci are in Hardy- Weinberg equilibrium. Linkage analysis may be performed alone, or in combination with direct detection of phenotypically evident polymorphisms. The use of microsatellite markers for genotyping is well documented. For examples, see Mansfield et al. (1994) Genomics 24:225-233; and Ziegle et al. (1992) Genomics 14:1026-1031. The use of SNPs for genotyping is illustrated in Underhill et al. (1996) Proc Natl Acad Sci U S A 93:196-200. Transgenic animals. The subject nucleic acids can be used to generate genetically modified non-human animals or site specific gene modifications in cell lines. The term "transgenic" is intended to encompass genetically modified animals having a deletion or other knock-out of UGT2B4, UGT2B7 or UGT2B15 activity, having an exogenous UGT2B4, UGT2B7 or UGT2B15 gene that is stably transmitted in the host cells, or having an exogenous UGT2B promoter operably linked to a reporter gene. Transgenic animals may be made through homologous recombination, where the UGT2B locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc. Genetically Modified Cells. Primary or cloned cells and cell lines are modified by the introduction of vectors comprising UGT2B4, UGT2B7 and UGT2B15 genetic polymorphisms. The gene may comprise one or more variant sequences, preferably a haplotype of commonly occurring combinations. In one embodiment of the invention, a panel of two or more genetically modified cell lines, each cell line comprising a UGT2B polymorphism, are provided for substrate and/or expression assays. The panel may further comprise cells genetically modified with other genetic sequences, including polymorphisms, particularly other sequences of interest for pharmacogenetic screening, e.g. UGT1, other UGT2 sequences, cytochrome oxidase polymorphisms, etc. Vectors useful for introduction of the gene include plasmids and viral vectors, e.g. retroviral-based vectors, adenovirus vectors, etc. that are maintained transiently or stably in mammalian cells. A wide variety of vectors can be employed for transfection and/or integration of the gene into the genome of the cells. Alternatively, micro-injection may be employed, fusion, or the like for introduction of genes into a suitable host cell. Genotyping Methods The effect of a polymorphism in the UGT2B4, UGT2B7 or UGT2B15 gene sequence on the response to a particular substrate or modifier is determined by in vitro or in vivo assays. Such assays may include monitoring the metabolism of a substrate during clinical trials to determine the UGT2B enzymatic activity, specificity or expression level. Generally, in vitro assays are useful in determining the direct effect of a particular polymorphism, while clinical studies will also detect an enzyme phenotype that is genetically linked to a polymorphism. The response of an individual to the substrate or modifier can then be predicted by determining the UGT2B genotype, with respect to the polymorphism. Where there is a differential distribution of a polymorphism by racial background, guidelines for drug administration can be generally tailored to a particular ethnic group. The basal expression level in different tissue may be determined by analysis of tissue samples from individuals typed for the presence or absence of a specific polymorphism. Any convenient method may be used, e.g. ELISA, RIA, ete. for protein quantitation, northern blot or other hybridization analysis, quantitative RT-PCR, etc. for mRNA quantitation. The tissue specific expression is correlated with the genotype. The alteration of UGT2B expression in response to a modifier is determined by administering or combining the candidate modifier with an expression system, e.g. animal, cell, in vitro transcription assay, etc. The effect of the modifier on UGT2B transcription and/or steady state mRNA levels is determined. As with the basal expression levels, tissue specific interactions are of interest. Correlations are made between the ability of an expression modifier to affect UGT2B activity, and the presence of the provided polymorphisms. A panel of different modifiers, cell types, etc. may be screened in order to determine the effect under a number of different conditions. A UGT2B polymorphism that results in altered enzyme activity or specificity is determined by a variety of assays known in the art. The enzyme may be tested for metabolism of a substrate in vitro, for example in defined buffer, or in cell or subcellular lysates, where the ability of a substrate to be metabolized by UGT2B4, UGT2B7 or UGT2B15 under physiologic conditions is determined. Where there are not significant issues of toxicity from the substrate or metabolite(s), in vivo human trials may be utilized, as previously described. The genotype of an individual is determined with respect to the provided UGT2B4, UGT2B7 and UGT2B15 polymorphisms. The genotype is useful for determining the presence of a phenotypically evident polymorphism, and for determining the linkage of a polymorphism to phenotypic change. A number of methods are available for analyzing nucleic acids for the presence of a specific sequence. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki et al. (1985) Science 230:1350-1354, and a review of current techniques may be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33. Amplification may be used to determine whether a polymorphism is present, by using a primer that is specific for the polymorphism. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al. (1990) Nucleic Acids Res 18:2887-2890; and Delahunty et al. (1996) Am J Hum Genet 58:1239-1246. A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), J'-dimethoxy^'.δ'- dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), radioactive labels, e.g. 3 P, 35 S, 3 H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product. The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, etc. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support, as described in U.S. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence of variant sequences. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), mismatch cleavage detection, and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP), the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels. In one embodiment of the invention, an array of oligonucleotides are provided, where discrete positions on the array are complementary to one or more of the provided polymorphic sequences, e.g. oligonucleotides of at least 12 nt, frequently 20 nt, or larger, and including the sequence flanking the polymorphic position. Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a different polymorphism. For examples of arrays, see Hacia et al. (1996) Nat Genet 14:441-447 and DeRisi et al. (1996) Nat Genet 14:457-460. The genotype information is used to predict the response of the individual to a particular UGT2B substrate or modifier. Where an expression modifier inhibits UGT2B expression, then drugs that are a UGT2B substrate will be metabolized more slowly if the modifier is co-administered. Where an expression modifier induces UGT2B expression, a co-administered substrate will typically be metabolized more rapidly. Similarly, changes in UGT2B activity will affect the metabolism of an administered drug. The pharmacokinetic effect of the interaction will depend on the metabolite that is produced, e.g. a prodrug is metabolized to an active form, a drug is metabolized to an inactive form, an environmental compound is metabolized to a toxin, etc. Consideration is given to the route of administration, drug-drug interactions, drug dosage, etc. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used- (e.g., amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric. EXAMPLE 1 Genotyping UGT2B4 Materials and Methods DNA Samples. Blood specimens from approximately 48 individuals were collected after obtaining informed consent. All samples were stripped of personal identifiers to maintain confidentiality. The only data associated with a given blood samples was gender and self-reported major racial group designations in the United States (Caucasian, Hispanic, African American). Genomic DNA was isolated from these samples using standard techniques. DNA was stored either as a concentrated solution, or in a dried form in microtiter plates. PCR amplifications. The primers used to amplify exons in which polymorphisms were found are shown in Table 1 , and were designed with NBI's Oligo version 5.1 program. Sequences for exons in which no polymorphisms were found are not shown. Table 1. UGT2B4 PCR Primers. Primar PCR Am lification Twenty-five nanograms of gDNA were amplified in the primary amplifications using the Perkin Elmer GeneAmp PCR kit according to the manufacturer's instructions in 25 μl reactions with AmpliTaq Gold DNA polymerase. Reactions contained 25 mM MgCI 2 and 0.2 μM of each primer. Thermal cycling was performed using a GeneAmp PCR System 9600 PCR machine (Perkin Elmer), utilizing a touch-down PCR protocol. The protocol, unless indicated otherwise in Table 2, consisted of an initial incubation of 95°C for 10 min, followed by ten cycles of 95°C for 20 sec, 64°C (minus 1°C per cycle) for 20 sec, 72°C for 2 min, six cycles of 95°C for 20 sec, 54°C for 20 sec, 72°C for 2 min, and nineteen cycles of 95°C for 20 sec, 54°C for 20 sec, 72°C for 2 min (plus 15 sec per cycle), and one final extension step of 72°C for 10 min. For the secondary PCR reactions, one microliter of each primary PCR reaction was re-amplified using the primary PCR primers. The thermal cycling profile that was used for the primary PCR for an exon was also used for the secondary PCR. Table 2. Cycling Profile Modifications Exon Primary PCR Secondary PCR 1 Touch-Down PCR step: 8 cycles same as Primary PCR 64° C (minus 1° C per cycle), for 15 sec Total Number of cycles: 35 4 Touch-Down PCR step: 10 cycles same as Primary PCR 64° C (minus 1° C per cycle), for 15 sec Total Number of cycles: 35 6 Touch-Down PCR step: 7 cycles same as Primary PCR 64° C (minus 1° C per cycle), for 15 sec Total Number of cycles: 35 DNA sequencing. PCR products from 48 individuals (approximately 1/3 African American, 1/3 Caucasian, 1/3 Hispanic) were prepared for sequencing by treating 8 μl of each PCR product with 0.15 μl of exonuclease I (1.5 U/reaction), 0.3 μl of Shrimp Alkaline Phosphatase (0.3U/reaction), q.s. to 10 μl with MilliQ water, and incubated at 37°C for 30 min, followed by 72°C for 15 min. Cycle sequencing was performed on the GeneAmp PCR System 9600 PCR machine (Perkin Elmer) using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's directions, with the following changes: (1) 2 μl of dRhodamine terminator premix, instead of 8 μl, (2) 10% (v/v) Dimethylsulfoxide was added to each individual nucleotide. The oligonucleotide primers (unlabelled), at 3 picomoles per reaction, used for the sequencing reactions are listed in Table 3. Sequencing reactions, with a final volume of 5 μl, were subjected to 25 cycles at 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min, followed by ethanol precipitation. After decanting the ethanol, samples were evaporated to dryness using a SpeedVac for roughly 15 min and were resuspended in 2 μl of loading buffer (5:1 deionized formamide:50 mM EDTA pH 8.0), heated to 94°C for 2 min, and were electrophoresed through 5.25% polyacrylamide/6M urea gels in an ABI Prism 377 DNA Sequencer, according to the manufacturer's instructions for sequence determination. All sequences were determined from both the 5' and 3' (sense and antisense) direction. Table 3. Sequencing Primers Table 4. Summar of UGT2B4 ol mor hisms. Nucleotide variants in exons are numbered from first base in Sequence 1. Amino Acid changes are numbered beginning with the first methionine in the protein sequence provided in Sequence 1. The nucleotide variant in intron 4 is numbered from the beginning of intron 4, as provided in Sequence 2.4. EXAMPLE 2 UGT2B7 Genotyping Twenty-five nanograms of gDNA were amplified in the primary amplifications using the Perkin Elmer GeneAmp PCR kit according to the manufacturer's instructions in 25 μl reactions with AmpliTaq Gold DNA polymerase. Reactions contained 25 mM MgCI 2 and 0.2 μM of each primer. Thermal cycling was performed using a GeneAmp PCR System 9600 PCR machine (Perkin Elmer), utilizing a touch-down PCR protocol. The exons for UGT2B7 were amplified using the following cycling conditions: An initial incubation at 96°C for 10 min., followed by 16 cycles of 95°C for 20 sec, 52°C for 20 sec, 72°C for 2 min., and nineteen cycles of 95°C for 20 sec, 52°C for 20 sec, 72°C for 2 min (plus 15 sec per cycle), and one final extension step of 72°C for 10 min. For the secondary PCR reactions, one microliter of each primary PCR reaction was re-amplified using the primary PCR primers. The thermal cycling profile that was used for the primary PCR for an exon was also used for the secondary PCR. The amplification primers are provided in Table 5, the sequencing primers in Table 6, and the polymorphisms in Table 7. Table 5 PCR Primers for UGT2B7 Am lification DNA sequencing. PCR products from 48 individuals were prepared for sequencing by treating 8 μL of each PCR product with 0.15 μL of exonuclease I (1.5U/reaction), 0.3 μL of Shrimp Alkaline Phosphatase (0.3U/reaction), q.s. to 10 μL with MilliQ water, and incubated at 37°C for 30 min, followed by 72 C C for 15 min. Cycle sequencing was performed on the GeneAmp PCR System 9600 PCR machine (Perkin Elmer) using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit or the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's directions, with the following changes: For the ABI Prism dRhodamine Terminator kit, (1) 2 μL of dRhodamine terminator premix, instead of 8 μL, (2) 10% (v/v) Dimethylsulfoxide was added to each individual nucleotide, (3) 5 μL total volume instead of 20 μL.. For the ABI Prism Big Dye Terminator kit (1) 0.8 μL of Big Dye terminator premix, instead of 8 μL, and (2) 15 μL total volume instead of 20 μL. The oligonucleotide primers (unlabeled), at 3 picomoles per reaction, used for the sequencing reactions are listed in Table 6. Sequencing reactions, with a final volume of 5 μL, were subjected to 25 cycles at 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min, followed by ethanol precipitation. After decanting the ethanol, samples were evaporated to dryness using a SpeedVac for roughly 15 min and were resuspended in 2 μl of loading buffer (5:1 deionized formamide:50 mM EDTA pH 8.0), heated to 94°C for 2 min, and were electrophoresed through 5.25% polyacrylamide/6M urea gels in an ABI Prism 377 DNA Sequencer, according to the manufacturer's instructions for sequence determination. All sequences were determined from both the 5' and 3' (sense and antisense) direction. Table 6 c Sequencing Primers I JGT2B7 P. No. F/R SEQ ID NO Primer Sequence 1, 2 F 66. Ggacataaccatgagaaatg R 67. Ttaagagcggatgagttgt 3,4 F 68. Tcatcatgcaacagattaag R 69. Cactacagggaaaaatagca 5 F 70. Accctttgtgtacagtctca R 71. Agctctgcttcaaagacac 6, 7 F 72. Ttgcctacattattctaaccc R 73. Ctttaccccacccattt 8, 9 F 74. Cattcctactctttatacagttctc R 75. Cccccgattcagactat 10 F 76. Cattcctactctttatacagttctc R 77. Cccccgattcagactat 11 , 12 F 78. Tcctccgaagtctgaaac R 79. Tataaaaaggatgaaactcacac 13 F 80. Tctgaggggttttgtctgta R 81. Ttttttgtctcaggaagaaaga 14 F 82. Aaaaaaagaaaaaaaaatcttttc R 83. Ccgcgatataagttcaacaa Example 3 Genotyping UGT2B15 Sequencing and analysis were performed as described in Example 2. The amplification primers are provided in Table 9, the sequencing primers in Table 8, and the polymorphisms in Table 10. Table 8 Se uencin Primers UGT2B15 Table 9 PCR Primers UGT2B15 All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims: What is Claimed is: 1. An isolated nucleic acid molecule comprising a UGT2B sequence polymorphism of SEQ ID NOs:25-38; 84-111 or 147-164, as part of other than a naturally occurring chromosome. 2. A nucleic acid probe for detection of UGT2B locus polymorphisms, comprising a polymorphic sequence of SEQ ID NOs:25-38; 84-111 or 147-164. 3. A nucleic acid probe according to Claim 2, wherein said probe is conjugated to a detectable marker. 4. An array of oligonucleotides comprising: two or more probes for detection of UGT2B locus polymorphisms, said probes comprising at least one form of a polymorphic sequences of SEQ ID NOs:25-38; 84-111 or 147-164. 5. A method for detecting in an individual a polymorphism in a UGT2B metabolism of a substrate, the method comprising: analyzing the genome of said individual for the presence of at least one UGT2B polymorphism of SEQ ID NOs:25-38; 84-111 or 147-164; wherein the presence of said predisposing polymorphism is indicative of an alteration in UGT2B expression or activity. 6. A method according to Claim 5, wherein said analyzing step comprises detection of specific binding between the genomic DNA of said individual with an array of oligonucleotides comprising: two or more probes for detection of UGT2B locus polymorphisms, said probes comprising at least one form of a polymorphic sequence of SEQ ID NOs:25-38; 84-111 or 147-164. 7. A method according to Claim 5, wherein said alteration in UGT2B expression is tissue specific. 8. A method according to Claim 5, wherein said alteration in UGT2B expression is in response to a UGT2B modifier. 9. A method according to Claim 8, wherein said modifier induces UGT2B expression. 10. A method according to Claim 8, wherein said modifier inhibits UGT2B expression.

Inventors: GALVIN MARGARET, MILLER ANDREW, PENNY LAURA, RIEDY MICHAEL, GALVIN, MARGARET, MILLER, ANDREW, PENNY, LAURA, RIEDY, MICHAEL
Applicants: AXYS PHARM INC, GALVIN MARGARET, MILLER ANDREW, PENNY LAURA, RIEDY MICHAEL, AXYS PHARMACEUTICALS, INC., GALVIN, MARGARET, MILLER, ANDREW, PENNY, LAURA, RIEDY, MICHAEL
Application year: 1999
Main classification: C12Q1
IPC codes: C12M1, C12N15, C12Q1