Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-20
2003-07-01
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024310, C435S091200
Reexamination Certificate
active
06586175
ABSTRACT:
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:1 14, 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: 1 5-24; 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
Galvin Margaret
Miller Andrew
Penny Laura
Riedy Michael
DNA Sciences Laboratories, Inc.
Goldberg Jeanine
Jones W. Gary
Sheridan & Ross P.C.
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