Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-17
2002-10-22
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06468744
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention resides in the field of molecular genetics and diagnostics.
2. Description of Related Art
Virtually all substances introduced into the human body (xenobiotics) as well as most endogenous compounds (endobiotics) undergo some form of biotransformation in order to be eliminated from the body. Many enzymes contribute to the phase I and phase II metabolic pathways responsible for this bioprocessing. Phase I enzymes include reductases, oxidases and hydrolases. Among the phase I enzymes are the cytochromes P450, a superfamily of hemoproteins involved in the oxidative metabolism of steroids, fatty adds, prostaglandins, leukotrienes, biogenic amines, pheromones, plant metabolites and chemical carcinogens as well as a large number of important drugs (Heim & Meyer,
Genomics
14, 49-58 (1992)). Phase II enzymes are primarily transferases responsible for transferring glucuronic acid, sulfate or glutathione to compounds already processed by phase I enzymes (Gonzales & Idle,
Clin. Pharmacokinet
. 26, 59-70 (1994)). Phase II enzymes include epoxide hydrolase, catalase, glutathione peroxidase, superoxide dismutase and glutathione S-transferase.
Many drugs are metabolized by biotransformation enzymes. For some drugs, metabolism occurs after the drug has exerted its desired effect, and result in detoxification of the drug and elimination of the drug from the body. Similarly, the biotransformation enzymes also have roles in detoxifying harmful environmental compounds. For other drugs, metabolism is required to convert the drug to an active state before the drug can exert its desired effect.
Genetic polymorphisms of cytochromes P450 and other biotransformation enzymes result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most human may cause intolerable side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug.
Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al.,
Toxicology Letters
64/65, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals is also important so that such individuals can be subjected to regular monitoring for the onset of cancers.
Existing methods of identifying deficiencies are not entirely satisfactory. Patient metabolic profiles are currently assessed with a bioassay after a probe drug administration. For example, a poor drug metabolizer with a CYP2D6 defect is identified by administering one of the probe drugs, debrisoquine, sparteine or dextromethorphan, then testing urine for the ratio of unmodified to modified drug. Poor metabolizers (PM) exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wildtype specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
The difficulties inherent in previous methods are overcome by the use of DNA chips to analyze mutations in biotransformation genes. The development of VLSIPS™ technology has provided methods for making very large arrays of oligonucleotide probes in very small areas. See U.S. Pat. No. 5,143,854, WO 90/15070 and WO 92/10092, each of which is incorporated herein by reference.
Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications. The present application describes the use of such chips for inter alia analysis of polymorphisms and copy number variations in genes of interest, particularly, biotransformation genes, such as cytochromes P450.
SUMMARY OF THE INVENTION
The invention provides methods for determining the copy number of a gene present in an individual. In such methods, a plurality of polymorphic sites from an individual are analyzed and the number of different polymorphic forms present at each site is thereby determined. Gene copy number is then assigned as the highest number of polymorphic forms present at a single site. Typically, the polymorphisms on in the gene whose copy number is being determined or in flanking sequences, although the polymorphism can be present elsewhere provided they are on the same chromosome as the gene whose copy number is being determined. To illustrate, if a single polymorphic form is present at each of the plurality of sites, the copy number of the gene is assigned as 1. If two polymorphic forms are present at one site and a single polymorphic form is present at each other of the plurality of sites, the copy number of the gene is assigned as 2. If three polymorphic forms are present at a first polymorphic site, a single polymorphic form is present at a second polymorphic site and two polymorphic forms are present at a third polymorphic site and the copy number of the gene is assigned as 3.
Often some or all of the polymorphisms analyzed are silent polymorphisms. Such silent polymorphisms can be present in a noncoding segment of the gene, such as an intronic segment, or in sequences flanking the gene. The more polymorphisms analyzed, the more likely one is to obtain an accurate result. Typically, analysis of about 10 or 50 polymorphisms is sufficient. Nucleic acids for analysis are typically prepared by obtaining a tissue sample from the individual containing the gene and amplifying the gene or a fragment thereof.
Polymorphisms are typically analyzed using probe arrays. Such analysis can be performed by contacting a nucleic acid comprising the gene or a fragment thereof with an array of oligonucleotides, the array comprising a plurality of subarrays, each subarray spanning a polymorphic site and complementarity to at least one polymorphic form of the gene at the site. Hybridization intensities of the nucleic acid to the oligonucleotides in the array are then detected. The pattern of hybridization indicates the number of polymorphic forms present at each polymorphic site. In some methods, subarrays are subdivided into probe groups, with different probe groups comprising probes complementary to different polymorphic forms at a site. In some methods, probe groups are subdivided into two or more probe sets. A first probe set comprises a plurality of probes spanning a polymorphic site of the gene, each probe comprising a segment of at least six nucleotides exactly complementary to a polymorphic form of the gene at the site, the segment including at least one interrogation position complementary to a corresponding nucleotide in the polymorphic form. A second probe set comprises a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at le
Chee Mark
Cronin Maureen T.
Fodor Stephen P. A.
Huang Xiaohua C.
Hubbell Earl A.
Affymetrix Inc.
Brusca John S.
Townsend and Townsend / and Crew LLP
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