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WO2002068448A1 - Procedes d'evaluation de la capacite de metabolisation de produits pharmaceutiques et de compositions de ceux-ci - Google Patents

Procedes d'evaluation de la capacite de metabolisation de produits pharmaceutiques et de compositions de ceux-ci Download PDF

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WO2002068448A1
WO2002068448A1 PCT/US2002/006135 US0206135W WO02068448A1 WO 2002068448 A1 WO2002068448 A1 WO 2002068448A1 US 0206135 W US0206135 W US 0206135W WO 02068448 A1 WO02068448 A1 WO 02068448A1
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nucleic acid
gene
acid molecule
sequence
gstml
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Inventor
Marco Guida
Jeff Hall
William P. Petros
James J. Vredenburgh
Oliver M. Colvin
Jeffrey R. Marks
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DNA Sciences Inc
Duke University
Cogenics Inc
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Genaissance Pharmaceuticals Inc
DNA Sciences Inc
Duke University
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Priority to CA002439403A priority Critical patent/CA2439403A1/fr
Publication of WO2002068448A1 publication Critical patent/WO2002068448A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to polymo ⁇ hisms in the cytochrome P450 3A4, cytochrome P450 3A5 and GSTMl genes and methods for genotyping and phenotyping individuals for such polymorphisms.
  • Cytochrome P450 enzymes are a heme-containing family that play central roles in oxidative, peroxidative and reductive metabolism of numerous endogenous and exogenous compounds, including many pharmaceutical agents. Substances known to be metabolized by P450 enzymes include steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines, retinoids, lipid hydroperoxides, phytoalexins, pharmaceuticals, environmental chemicals and pollutants. P450 substrates also include natural plant products involved in flavor, odor and flower color.
  • P450 enzymes also have functions in maintaining steady-state levels of endogenous ligands involved in ligand-modulated transcription of genes effecting growth, apoptosis, differentiation, cellular homeostasis, and neuroendocrine functions.
  • the metabolism of foreign chemicals by P450 enzymes can produce toxic metabolites, some of which have been implicated as agents responsible for birth defects and tumor initiation and progression.
  • the P450 gene superfamily is likely to have evolved from an ancestral gene present before the prokaryote/eukaryote divergence.
  • the number of individual P450 genes in any mammalian species is estimated at 60 to 200.
  • the cytochrome P450 (CYP) 3A subfamily is unique in that it is present in large amounts in human liver microsomes, and there are many forms in the subfamily.
  • CYP3A4 cytochrome P450
  • CYP3A4 and CYP3A7 genes are 87% homologous by amino acid and 95% homologous by nucleotide sequence, while CYP3A4 and CYP3A5 are only 88% homologous in the coding region.
  • CYP3A4 and CYP3A7 are 91% homologous in the 5'-flanking sequences, differing by the presence of a unique P450NF specific element (NFSE) and a P450HFLa specific element (HFLaSE), respectively (Hashimoto et al, 1993).
  • NFSE unique P450NF specific element
  • HLaSE P450HFLa specific element
  • cytochrome P450 enzymes result in subpopulations of individuals that are distinct in their ability to perform particular drug biotransformation reactions. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to the majority of humans may cause intolerable side-effects in an individual suffering from a defect in a cytochrome P450 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 the lack of a particular cytochrome P450 enzyme required for conversion of the drug to a metabolically active form.
  • the CYP3A subclass catalyzes a remarkable number of oxidation reactions of clinically important drugs such as quinidine, warfarin, erythromycin, cyclosporin A, midazolam, lidocain, nifedipine, and dapsone.
  • clinically important drugs such as quinidine, warfarin, erythromycin, cyclosporin A, midazolam, lidocain, nifedipine, and dapsone.
  • Current estimates are that more than 60% of clinically used drugs are metabolized by the CYP3A4 enzyme, including such major drug classes as calcium channel blockers, immunosuppressors, macrolide antibiotics and anticancer drugs, see Brian et al., 1990, Biochemistry, vol. 29, pages 11280-11292.
  • CYP3A4 is expressed in all adult human liver and intestine, accounting for more than 50% of total P450 in both organs. Expression is inducible in vivo and in vitro by numerous compounds such as rifampicin, barbiturates and glucocorticoids. In kidney, CYP3A4 is expressed polymo ⁇ hically. CYP3A4 expression is sex-influenced, as females have 24% greater expression than males. Substantial inter-individual variation in the metabolism of specific compounds by CYP3 A4 has been reported (Kleinbloesem et al., Biochemical Pharmacology, 1984, vol. 33, pages 3721-3724. U.S. Patent No.
  • CYP3A4 variant associated with a heightened risk of developing or having prostate cancer and a decreased risk for developing treatment-related leukemias.
  • the polymo ⁇ hism disclosed by Rebbeck et al. is an A to G transition in the promoter region of the CYP 3A4 gene which is thought to alter the nifedipine-specific binding element located 287 to 296 bases 5' to the CYP 3A4 transcription start site.
  • the genotype associated with this variant is believed to increase the production of potentially DNA damaging reactive intermediates upon patient exposure to an epipodophyllotoxin.
  • CYP3A5 is detected in 10-30% of Caucasian adult livers, and expressed constitutively in adult kidney.
  • CYP3A5 expression does not appear to be sex-influenced and only moderately inducible by xenobiotics both in vivo and in vitro.
  • CYP3A7 is expressed in fetal liver but only in 25% of adult livers. Molecular mechanisms responsible for the developmentally specific expression of CYP3 A's are unknown.
  • GST glutathione S-transferase
  • GST alleles associated with impaired detoxification will confer an increased susceptibility to a wide range of diseases.
  • GST genotypes have been associated with an increased susceptibility in diseases associated with oxidative stress.
  • null mutation in the mu class GSTMl gene is polymo ⁇ hic due to large deletions in the structural gene.
  • the null GSTMl genotype is clearly associated with bladder cancer and lung cancer, and possibly associated with colorectal, hepatocellular, gastric, esophageal, head and neck as well as cutaneous cancer. There is considerable evidence that the combination of the GSTMl null genotype in combination with the cytochrome P450 1 Al rare alleles confers a highly increased risk of developing lung cancers in smokers.
  • the GSTMl null genotype has also been found to be significantly associated with an increased risk of developing postmenopausal breast cancer as described by Helzlsouer et al, J. Natl. Cancer Inst, 1998, vol. 90, pages 512-518. Women have a 15 percent lifetime risk of developing breast cancer.
  • Available treatment options include ( 1 ) surgery, (2) radiation therapy, (3) chemotherapy, and (4) hormone manipulation.
  • Two drugs commonly used in the treatment of breast cancer are cyclophosphamide and carmustine (BCNU).
  • Cyclophosphamide is a nitrogen mustard derivative, polyfunctional alkylating agent which is bioconverted from an inert prodrug to an active DNA alkylating agent by the oxidative cytochromes of the liver of which CYP 3A4 and 3A5 are the principle enzymes.
  • CYP 3A4 and 3A5 are the principle enzymes.
  • Cyclophospamide functions to interfere with DNA replication and transcription of RNA, ultimately resulting in the disruption of nucleic acid function.
  • the drug also exhibits potent immunosuppressive activity and phosphorylating properties that enhance its cytotoxicity.
  • cyclophosphamide used alone has been reported to produce objective responses in about 35% of patients. Used in combination regimens, objective responses have been reported in up to 90% of patients, and cyclophosphamide-containing combinations are believed by some experts to be the treatment of choice.
  • BCNU is a nitrosourea with a broad spectrum of activity. It is a classic alkylating agent, but also inhibits DNA repair by isocyanate formation. BCNU is used alone or as a component of various chemotherapy regimens in the treatment of primary or metastatic tumors. BCNU is a highly toxic drug with a low therapeutic index, thus a therapeutic response is unlikely without some evidence of toxicity. The primary toxicities are pulmonary toxicity and hepatic dysfunction which appear to be dose related. Patients receiving cumulative doses exceeding 1400mg/m 2 are at substantially higher risk than patients receiving lower cumulative doses. Thus, any means of screening prospective cancer patients for factors which can effect the dosing of BCNU is of great importance in effectively designing chemotherapy regimens that include BCNU to enhance the clinical outcome while minimizing adverse effects.
  • Cisplatin is a bifunctional alkylating agent that binds to DNA and inhibits DNA synthesis.
  • the drug produces predominately DNA interstrand crosslinks with some intrastrand crosslinks resulting from the formation of adducts between activated platinum complexes of the drug. Interstrand crosslinking appears to correlate well with the cytotoxicity of the drug.
  • Cisplatin is used to treat a wide variety of neoplasms and is often used as a component of combination chemotherapeutic regimens because of its relative lack of hematologic toxicity. Cisplatin is a highly toxic drug with a low therapeutic index.
  • hematologic toxicities such as thrombocytopenia and leukopenia are the major dose-limiting adverse effects of cisplatin therapy
  • other dose-limiting adverse effects including nephrotoxicity, ototoxicity, neurotoxicity, and emesis are frequently seen.
  • nephrotoxicity, ototoxicity, neurotoxicity, and emesis are frequently seen.
  • nephorotoxic or ototoxic effects such as aminoglycoside antibiotics.
  • the present invention is directed to novel polymo ⁇ hisms in the human CYP3 A4 and CYP3A5 genes and the detection of polymo ⁇ hisms in genes encoding the CYP3A4, CYP3A5 and GSTMl metabolic enzymes in individuals.
  • the polymo ⁇ hisms detected can influence the outcome of breast cancer treatments and the selection of chemotherapeutic agents used to treat breast cancer in the patients tested.
  • These polymo ⁇ hisms are predictive of altered metabolism of chemotherapeutic agents used in the treatment of breast cancer and/or the occurrence of disease.
  • CYP3 A4 and CYP3 A5 polymo ⁇ hic nucleic acid sequences and methods to use such nucleic acid sequences and polymo ⁇ hic GSTMl sequences, in particular for diagnostic pu ⁇ oses to identify individuals having a polymo ⁇ hic genotype.
  • the present invention is directed to an isolated nucleic acid molecule, comprising a sequence selected from the sequence of SEQ ID NO: l; and the sequence fully complementary thereto.
  • the present invention is also directed to an isolated nucleic acid molecule which includes at least one base variation from that of the human CYP3A4 sequence, wherein the nucleic acid molecule is selected from a nucleic acid molecule that comprises a G nucleotide for an A nucleotide at position -392 of the promoter of said CYP3A4 gene with respect to the start codon of said CYP3A4 gene and at least 17 other bases of said CYP3A4 gene contiguously appurtenant thereto, and a nucleic acid molecule which is fully complementary thereto.
  • the nucleic acid molecule can include a sequence selected from the group consisting of SEQ ID NO: 1 , and/or a nucleic acid sequence which is fully complementary to SEQ ID NO: 1.
  • the present invention is directed to an isolated nucleic acid molecule, comprising a sequence selected from the sequence of SEQ ID NO:2; and the sequence fully complementary thereto.
  • the present invention is also directed to an isolated nucleic acid molecule which includes at least one base variation from that of the human CYP3A5 sequence, wherein the nucleic acid molecule is selected from a nucleic acid molecule that comprises a G nucleotide for an A nucleotide at position -147 of the promoter of said CYP3 A5 gene with respect to the start codon of said CYP3 A4 gene and at least 33 other bases of said CYP3A5 gene contiguously appurtenant thereto, and a nucleic acid molecule which is fully complementary thereto.
  • the nucleic acid molecule can include a sequence selected from the group consisting of SEQ ID NO:2 and/or a nucleic acid sequence which is fully complementary to SEQ ID NO:2.
  • a further embodiment of the present invention includes a method of detecting a variant gene having a polymo ⁇ hism associated with reduced metabolism of a substrate selected from the group consisting of a CYP3A4 substrate, a CYP3A5 substrate and a GSTMl substrate in an individual. This method includes obtaining a nucleic acid sample comprising a gene isolated from the individual, the gene being selected from a CYP3A4 gene, a CYP3A5 gene and a GSTMl gene.
  • the method further includes detecting the presence or absence in the individual of a polymo ⁇ hism selected from (i) a substitution of a G nucleotide for an A nucleotide at position -392 of the promoter of the CYP3 A4 gene with respect to the start codon of the CYP3A4 gene, wherein the presence of the substitution is associated with reduced CYP3 A4 substrate metabolism: (ii) a substitution of a G nucleotide for an A nucleotide at position -147 of the promoter of the CYP3A5 gene, wherein the presence of the substitution is associated with reduced CYP3A5 substrate metabolism: and (iii) a GSTMl null mutation, wherein the presence of the GSTMl null mutation is associated with reduced GSTMl substrate metabolism.
  • the method can also include determining whether the individual is homozygous or heterozygous for the polymo ⁇ hism.
  • a variety of assays are suitable for determining whether any of the identified variant sequences are present.
  • the substrates in question for any one of the three genes can be selected from cyclophosphamide and BCNU.
  • the present invention includes a method for selecting a treatment for a cancer patient.
  • the method the genotyping steps described above and further includes selecting a cancer treatment regime that does not include administration of an anti- cancer agent selected from the group consisting of cyclophosphamide and BCNU if one or more of the polymo ⁇ hisms are present.
  • a further method for selecting a treatment for a cancer patient can include selecting a cancer treatment regime that includes administration of an anti-cancer agent selected from the group consisting of cyclophosphamide and BCNU if none of the polymo ⁇ hisms are present.
  • the present invention relates to nucleic acid molecule compositions that contain certain genetic characteristics and methods that identify the presence or absence of such characteristics in patients.
  • the present invention includes the identification of different genetic polymo ⁇ hisms in the cytochrome P4503A4, cytochrome P4503A5 and the GSTMl genes. The presence or absence of certain polymo ⁇ hisms in one or more of these genes has been found to be prognostic for a therapeutic response to certain antineoplastic agents.
  • a construct includes a plurality of such constructs and reference to “the CYP3 A4 nucleic acid” includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth.
  • an "isolated nucleic acid molecule” refers to a nucleic acid molecule which is the size of or smaller than a gene. Thus, an isolated nucleic acid molecule does not encompass isolated genomic DNA or an isolated chromosome. The term isolated nucleic acid molecule does not connote any specific minimum length. It should also be appreciated that reference to an isolated nucleic acid molecule does not necessarily reflect the extent of purity of the nucleic acid molecule.
  • An isolated nucleic acid molecule of the present invention can be obtained from a natural source, such as a tissue sample, or it can be produced using molecular biology techniques, such as by PCR amplification, or it can be produced by chemical synthesis.
  • allelic has the meaning which is commonly known in the art, that is, a genomic variant of a referent gene, including variants, which, when translated result in functional or dysfunctional (including non-existant) gene products.
  • the first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form.
  • “Contiguously appurtenant to” means any bases flanking the referent position, including the instances of all bases selected 5' to the referent position and no bases selected 3' to the referent position; all bases selected 3' to the referent position and no bases selected 5' to the referent position; and some bases selected 5' and some bases selected 3' to the referent position.
  • the term is intended to mean that the selected bases necessarily must be in the same sequential order as described in the referent sequence, with the exception of the variant base at the referent position.
  • "For the pu ⁇ ose of determining genotype” means that one of the pu ⁇ oses is to determine genotype, not necessarily that the end goal or use of the information is to determine genotype. For instance, "for the pu ⁇ ose of determining genotype” includes the use of the information to determine genotype for the ultimate goal of determining probability of negative or positive drug interactions.
  • Gene has the meaning that is commonly-known in the art, that is, a nucleic acid sequence that includes the translated sequences that code for a protein ("exons”) and the untranslated intervening sequences ("introns”), and any regulatory elements ordinarily necessary to translate the protein.
  • Genetype has the meaning that is commonly-known in the art, that is, a physical description of a nucleic acid sequence.
  • Hybridization has the meaning that is commonly-known in the art, that is, the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain some regions of mismatch.
  • Polymo ⁇ hism means a polymo ⁇ hism wherein the group exists by virtue of a difference in identity of one or more nucleotides at given sequence locations. The location of nucleotide identity differences is usually preceded by and followed by highly conserved sequences (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). However, more than one single nucleotide polymo ⁇ hism can exist between or among the group members.
  • a “transition” is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a “transversion” is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymo ⁇ hisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a given sequence location.
  • “Stringent hybridization” means that which is commonly-known in the art, that is, at a salt concentration of no more than 1M and a temperature of at least 25 degrees Celsius. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM Sodium Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 55 degrees to 60 degrees Celsius are suitable.
  • “Substrate” means a chemical entity that is modified by activity of a CYP3A4, CYP3A5 and/or GSTMl enzyme, usually under normal physiological conditions. Most of these substrates are lipophilic compounds.
  • a substrate of any one or more of CYP3A4, CYP3A5 and GSTMl is selected from the group consisting of cyclophosphamide, cisplatin and BCNU.
  • a substrate of any one or more of CYP3A4, CYP3A5 and GSTMl is selected from the group consisting of cyclophosphamide and BCNU.
  • alleles are expressed by symbols in accordance with definitions given by IUPAC-IUB and common names or common usage in the art.
  • One embodiment of the present invention is an isolated nucleic acid molecule comprising a CYP3 A4 sequence polymo ⁇ hism of SEQ ID NO: 1 as part of other than a naturally occurring chromosome, as described in detail below.
  • Another embodiment of the present invention is an isolated nucleic acid molecule comprising a CYP3 A5 sequence polymo ⁇ hism of SEQ ID NO:2 as part of the other than a naturally occurring chromosome.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid molecule of the present invention can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • the nucleic acid molecules of the present invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acid molecule will be obtained substantially free of other nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably.
  • the nucleic acid sequence of the CYP3A4 gene is known in the art and accessible in public databases. Genbank Accession No. D 11131 provides a partial sequence of the human cytochrome P450 3A4 gene. Genbank Accession No. Ml 8907 (cDNA) provides the cDNA sequence of a human CYP3A4 allele.
  • the promoter region of the CYP3 A4 gene is provided as SEQ ID NO:3.
  • the nucleic acid sequence of the CYP3A5 gene is generally known in the art and accessible in public databases.
  • Genbank Accession No. S74699 provides the CYP3 A5 5' genomic region.
  • the present inventors have sequenced a portion of the promoter region and exon 1 of the CYP3 A5 gene and found errors in Genbank Accession No. S74699.
  • the correct sequence for the portion of the promoter and exon 1 is provided as SEQ ID NO:4.
  • the nucleic acid sequence of the GSTMl gene is known in the art and accessible in public databases.
  • the null mutation in the mu class GSTMl gene is well known in the art. For example, see Xu, S.; Wang, Y.; Roe, B.; Pearson, W. R. : Characterization of the human class mu glutathione S-transferase gene cluster and the GSTMl deletion. J. Biol. Chem.273: 3517-3527, 1998.
  • CYP3A4 gene As used herein, the term "CYP3A4 gene,” “CYP3A5 gene” or “GSTMl gene” is intended to refer to both the wildtype and polymo ⁇ hic sequences, unless specifically denoted otherwise. Nucleic acids of particular interest comprise the provided polymo ⁇ hic sequences. It is within the skill of one in the art to identify the location of a polymo ⁇ hic sequence of the present invention using wildtype CYP3A4, CYP3A5 or GSTMl genomic or cDNA sequences known in the art.
  • a skilled artisan can use a polymo ⁇ hic sequence, its corresponding wildtype sequence and the CYP3A4, CYP3A5 or GSTMl sequence contiguously appertanant to the referenced polymo ⁇ hism to determine the position of the polymo ⁇ hism.
  • the positions of the CYP3A4 and CYP3A5 polymo ⁇ hisms of the present inventions are described below in Table 1. TABLE 1
  • the polymo ⁇ hism of the CYP3 A4 sequence occurs at nucleotide -392 of the promotor region of the CYP3A4 genomic sequence. Sequences in the promotor region are denoted by negative numbers corresponding to the number of nucleotides 5' to the start codon.
  • the start codon of the CYP3A4 gene is at nucleotides 1208-1210 of SEQ ID NO:3.
  • the polymo ⁇ hism is a base pair substitution of A to G.
  • the polymo ⁇ hism is in the promoter region so it does not result in an amino acid substitution.
  • CYP3A4 polymo ⁇ hism is described in detail in WO 9913106A1, published March 18, 1999 and is identified in SEQ ID NO:44 therein.
  • the CYP3A4 polymo ⁇ hism can be identified as an A to G transition at position -290 with reference to the transcription initiation site. More particularly, the CYP3 A4 polymo ⁇ hism occurs at nucleotide 816 of SEQ ID NO:3.
  • One embodiment of the present invention is an isolated nucleic acid molecule that comprises the foregoing human cytochrome P450 3A4 polymo ⁇ hism.
  • Such an isolated nucleic acid molecule includes the polymo ⁇ hism and at least 17 other bases, alternatively at least 20 other bases, at least 30 other bases, at least 40 other bases, or at least 50 other bases of the wildtype sequence contiguously appurtenant thereto.
  • the present invention includes human CYP3A4 alleles that comprise the CYP3 A4 polymo ⁇ hism as described herein, having appurtenant sequences of 10, 15, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 250, 300, 350, 400, 450, 500, or 1000 bases, or any whole number encompassed by the range of 1-10,000.
  • Further embodiments of the present invention include nucleic acid molecules having a sequence fully complementary to any of the sequences described above.
  • the polymo ⁇ hism of the CYP3 A5 sequence occurs at nucleotide -147 of the promotor region of the CYP3A5 genomic sequence. Sequences in the promotor region are denoted by negative numbers corresponding to the number of nucleotides 5' to the start codon.
  • the start codon of the CYP3A5 gene is at nucleotides 1184-1186 of SEQ ID NO:4.
  • the polymo ⁇ hism is a base pair substitution of A to G.
  • the polymo ⁇ hism is in the promoter region so it does not result in an amino acid substitution.
  • the CYP3A5 polymo ⁇ hism can be identified as an A to G transition at position -44 with reference to the transcription initiation site. More particularly, the CYP3A5 polymo ⁇ hism occurs at nucleotidel037 of SEQ ID NO:4.
  • One embodiment of the present invention is an isolated nucleic acid molecule that comprises the foregoing human cytochrome P450 3A5 polymo ⁇ hism. Such an isolated nucleic acid molecule includes the polymo ⁇ hism and at least 33 other bases, alternatively at least 40 other bases, or at least 50 other bases of the wildtype sequence contiguously appurtenant thereto.
  • the present invention includes human CYP3A5 alleles that comprise the CYP3A5 polymo ⁇ hism as described herein, having appurtenant sequences of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 250, 300, 350, 400, 450, 500, or 1000 bases, or any whole number encompassed by the range of 1 -10,000.
  • Further embodiments of the present invention include nucleic acid molecules having a sequence fully complementary to any of the sequences described above.
  • the polymo ⁇ hism of the GSTMl gene is the well-characterized null mutation.
  • the genotype of an individual is determined with respect to the provided CYP3A4, CYP3A5 and/or GSTMl gene polymo ⁇ hisms.
  • the genotype is useful for determining the presence of phenotypically evident polymo ⁇ hism having the effect of reduced metabolism of a CYP3A4, CYP3A5 or GSTMl substrate, respectively.
  • One embodiment of the present invention is a method of identifying a sample containing a nucleic acid molecule that comprises a wildtype or variant allele, the method comprising identifying the presence or absence of one or more of the CYP3A4, CYP3 A5 or GSTMl polymo ⁇ hisms described herein.
  • Many of the diagnostic assays rely on amplification of part or all of a CYP3A4, CYP3A5 and/or GSTMl nucleic acid molecule.
  • portions of a nucleic acid molecule are amplified by the polymerase chain reaction (PCR). The PCR process is described in e.g., U.S. Pat. Nos.
  • nucleic acid molecules in accordance with the present invention In a sample by PCR, the sequence must be accessible to the components of the amplification system. Accessibility can be achieved by isolating nucleic acid molecules from the sample. A variety of techniques for extracting nucleic acid molecules from biological samples are known in the art. Alternatively, if the sample is fairly readily disruptable, the nucleic acid need not be purified prior to amplification by the PCR technique, i.e., if the sample comprises cells, particularly peripheral blood lymphocytes or monocytes, lysis and dispersion of the intracellular components may be accomplished merely by suspending the cells in hypotonic buffer. See Han et al., Biochemistry, 1987, vol.
  • Polymo ⁇ hisms are detected in a nucleic acid molecule from an individual being analyzed.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable. Examples of convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • Nucleic acid molecules can be obtained according to procedures well-known in the art.
  • a first step is the synthesis of a DNA copy (cDNA) of the region to be amplified by reverse transcription.
  • Reverse transcription is the polymerization of deoxynucleoside triphosphates to form primer extension products that are complementary to a ribonucleic acid template.
  • the process is effected by reverse transcriptase, an enzyme that initiates synthesis at the 3 '-end of the primer and proceeds toward the 5'-end of the template until synthesis terminates.
  • suitable polymerizing agents that convert the RNA nucleic acid molecule into a complementary, cDNA sequence are avian myeloblastosis virus reverse transcriptase and Thermus thermophilous DNA polymerase.
  • Reverse transcription can be carried out as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR).
  • Polymerizing agents suitable for synthesizing a cDNA sequence from the RNA template are reverse transcriptase (RT), such as avianmyeloblastosis virus RT, Moloneymurine leukemia virus RT, or Thermus thermophilous DNA polymerase.
  • Primers for PCR amplification are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer.
  • the primers are selected to be substantially complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer with the remainder of the primer sequence being complementary to the strand.
  • complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer.
  • Paired primers for amplification of a given segment of DNA are designated forward and reverse primers.
  • the forward primer hybridizes to a double-stranded DNA molecule at a position 5', or upstream, from the reverse primer.
  • the forward primer hybridizes to the complement of the coding strand of the double stranded sequence, i.e., the antisense strand, and the reverse primer hybridizes to the coding strand.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from about 10 to about 100, preferably about 15 to about 50, more preferably about 15 to about 30, or more preferably about 15 to about 25 nucleotides in length.
  • the spacing of primers determines the length of segment to be amplified. The spacing is not usually critical and amplified segments can range in size from about 25 bases to at least about 35 kilobases in length. Segments from about 25 to about 2000, preferably about 50 to about 1000, more preferably about 100 to about 500 nucleotides in length are typical.
  • a primer can be labeled, if desired, by inco ⁇ orating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in an ELISA), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • a label can also be used to "capture" the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • An allele-specific primer can be used in a PCR amplification.
  • the allele-specif ⁇ c primer hybridizes to a site on a nucleic acid molecule that overlaps with a polymo ⁇ hism and extension will only occur if an allelic form complementary to the primer is present. See Gibbs, Nucleic Acid Res., 1989, vol. 17, pages 2427-2448.
  • This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. Thus, the presence or absence of an amplification product is detected using standard methods.
  • Controls can be used that test the efficacy of the amplification reaction itself or that allow the experimental results to be compared with known wildtype or polymo ⁇ hic CYP3 A4, CYP3A5 and/or GSTMl nucleic acid molecule samples.
  • the method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymo ⁇ hism because this position is most destabilizing to elongation from the primer.
  • sample nucleic acid molecules isolated directly from cells, amplified or cloned fragments, can also be analyzed by a number of other methods known in the art.
  • the nucleic acid molecule can be sequenced by using either the dideoxy chain termination method or other methods (see for example Sambrook et al. , Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).
  • Hybridization using allele-specific probes can be used to determine the presence or absence of a polymo ⁇ hism by, for example Southern blot, dot blots, etc.
  • An allele-specific probe can be designed that hybridizes to a segment of a nucleic acid molecule from one individual but does not hybridize to the corresponding segment from another individual due to the presence of different polymo ⁇ hic forms in the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles.
  • hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support can also be used as a means of detecting the presence of variant sequences.
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis (DGGE). Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and
  • Alleles of target sequences can be differentiated using single-strand conformation polymo ⁇ hism analysis (SSCP), which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al. , Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single- stranded amplification products can be related to base-sequence difference between alleles of target sequences.
  • Other methods of detection include mismatch cleavage detection and heteroduplex analysis in gel matrices. These methods are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • a polymo ⁇ hism creates or destroys a recognition site for a restriction endonuclease, referred to as restriction length polymo ⁇ hism, or 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.
  • an array of oligonucleotides are provided, where discrete positions on the array are complementary to one or more of the provided polymo ⁇ hic sequences, e.g. oligonulcoetides of at least 12 nucleotides, frequently 20 nucleotides or larger and including the sequence flanking the polymo ⁇ hic position.
  • Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a different polymo ⁇ hism.
  • Arrays of interest may further comprise sequences, including polymo ⁇ hisms, of other genetic sequences, particularly other sequences of interest for pharmacogenetic screening.
  • one or more CYP3A4, CYP3A5 and GSTMl polymo ⁇ hisms provided herein can be detected in a single assay such as a multiplex assay to identify the presence or absence of different alleles in the same assay, see for example Stuven et al, Pharmacogenetics, 1996, vol. 6, pages 417-421.
  • a polymo ⁇ hism provided herein is indicative of a poor clinical outcome of breast cancer treatment using cyclophosphamide, BCNU or combinations of these drugs together or in combination with other chemotherapeutic regimens.
  • a preferred strategy for analysis entails amplification of a DNA sequence spanning the polymo ⁇ hism, in the instance of CYP3A4 or CYP3A5 polymo ⁇ hisms of the present invention.
  • Amplification of such a sequence can be primed from forward and reverse primers that hybridize to a CYP3 A4or CYP3 A5 gene on opposite sides of the polymo ⁇ hism but which do not hybridize to the variant nucleotide sequence itself. That is, for detection of the polymo ⁇ hism, the forward primer hybridizes upstream or 5' to the polymo ⁇ hic site and the reverse primer hybridizes downstream or 3' to this site.
  • the forward primer is sufficiently complementary to the antisense strand of a nucleic acid molecule to hybridize therewith and the reverse primer is sufficiently complementary to the sense strand of the sequence to hybridize therewith.
  • the primers usually comprise first and second subsequences from opposite strands of a double-stranded DNA sequence. It is particularly important to avoid mismatches in the two nucleotides at the 3' end of the primer (especially the terminal nucleotide). Having amplified a segment of a gene known to span a polymo ⁇ hism, a variety of assays are available for determining whether the polymo ⁇ hism is present that are disclosed herein, preferably, using allele specific primers.
  • selective amplification of the wildtype allele of the gene in question allele can be accomplished using a forward primer that has about 10-50, and usually 15-30 nucleotides from the wildtype genomic sequence, including the portion of the gene corresponding to the position of the polymo ⁇ hism.
  • a forward primer when paired with any suitable reverse primer downstream from the polymo ⁇ hic nucleotide sequence (i.e., sufficiently complementary to the sense strand of gene to hybridize therewith) can be used to amplify selectively the wildtype allele without amplifying a mutant allele.
  • the polymo ⁇ hic nucleotide sequence usually occurs near, or preferably at, the 3' end of the primer.
  • a reverse primer that has about 10-50 or usually 15-30 contiguous nucleotides from the complement of the wildtype genomic sequence (i.e., the antisense strand) including the polymo ⁇ hic nucleotide sequence.
  • a reverse primer can be paired with any suitable forward primer sufficiently complementary to a sequence of the antisense strand of the gene upstream from the polymo ⁇ hic nucleotide sequence to hybridize therewith.
  • the position corresponding to the polymo ⁇ hic nucleotide sequence should again be at or near the 3' end of the reverse primer.
  • a suitable forward primer for amplification comprises about 10-50 or usually 15-30 contiguous nucleotides including a polymo ⁇ hic nucleotide sequence from the mutant genomic sequence (i.e., the sense strand).
  • the forward primer can be paired with any suitable reverse primer sufficiently complementary to the sense strand of a genomic subsequence downstream from the polymo ⁇ hic nucleotide sequence to hybridize therewith.
  • the same result can be achieved using a reverse primer comprising about 10-50 or 15-30 contiguous nucleotides including the polymo ⁇ hic nucleotide sequence from the complement of the mutant sequence (i.e., the antisense strand).
  • Such a reverse primer can be paired with any suitable forward primer sufficiently complementary to the antisense strand of a subsequence upstream from a polymo ⁇ hic nucleotide sequence to hybridize therewith.
  • the sample under test is characterized as wildtype or mutant by the presence or absence of an amplification product.
  • primer designed for selective amplification of the wildtype allele the presence of an amplification product is indicative of that allele and the absence of an amplification product indicative of a mutant allele.
  • primers designed for selective amplification of a mutant allele are examples of primers designed for selective amplification of a mutant allele.
  • a sample is divided into two aliquots, one of which is amplified using primers for wildtype allele amplification, the other of which is amplified using primers appropriate for mutant allele amplification.
  • the presence of an amplification product in one but not both of the aliquots indicates that the individual under test is either wildtype or a homozygous for the mutation (depending on aliquot in which the amplification product occurred).
  • the presence of amplification product in both aliquots indicates that the individual is heterozygous.
  • the absence of an amplification product in both aliquots would indicate either the absence of a gene or a quality control problem in the amplification procedure requiring that the assay be repeated.
  • the presence or absence of amplification products can be detected by gel electrophoresis using methods standard in the art or described herein.
  • a preferred strategy for analysis entails amplification using any portion of sequence in the coding region of the GSTMl gene that would produce a detectable PCR product if the gene is wildtype, but no PCR product if the null mutation is present.
  • some conventional methods for determining hetero- or homozygosity such as that described above, would not be useful for the GSTMl null mutation, those skilled in the art could design suitable methods. For example, one could conduct an assay in which two differently sized PCR amplification products would be produced for a heterozygote. Such an assay could be designed once the breakpoints for the null mutation were determined.
  • the various genotyping methods described herein can be used in a method for selecting a treatment for a cancer patient.
  • the polymo ⁇ hisms of the present invention have been associated with reduced metabolism of various chemotherapeutic agents, specifically cyclophosphamide and BCNU.
  • a cancer treatment regime is selected to account for the phenotypic result of the identified genotype.
  • a cancer treatment regime can be selected that does not include administration of an anti-cancer agent that is metabolized by an expression product of one of the genes described herein, and particularly, cyclophosphamide and BCNU.
  • a cancer treatment regime can be selected that includes administering an anti-cancer agent that is metabolized by an expression product of one of the genes described herein at higher than conventional doses. More particularly, such doses can be at least about 1% higher than conventional doses, more preferably, at least about 10 % higher than conventional doses, and even more preferably, at least about 50 % higher than conventional doses. Conventional doses of known chemotherapeutic agents are well-known to those of skill in the art.
  • conventional doses of cyclophosphamide can include 40-100 mg/Kg.
  • Conventional doses of BCNU can include 100-200 mg/m 2 every 6 weeks.
  • a cancer treatment regime that includes administration of an anti-cancer agent selected from the group consisting of cyclophosphamide and BCNU is selected. More particularly, this method includes administration of one of such anti-cancer agents at conventional doses.
  • kits comprises useful components for practicing the methods of the present invention.
  • the kit typically comprises at least one of the primers needed for the PCR amplification if PCR amplification is used and also control DNA suitable for determining the success of the PCR reaction and/or to confirm the identification of the presence or absence of a polymo ⁇ hism in a sample.
  • a kit usually contains a matched pair of forward and reverse primers as described above for amplifying a segment encompassing a polymo ⁇ hism of the present invention.
  • kits usually contain a pair of primers for amplification of the mutant allele and/or a separate pair of primers for amplification of the wildtype allele.
  • kits include, for example, restriction enzymes for analysis of amplification products, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, and the appropriate buffers for reverse transcription, PCR, or restriction enzyme reactions.
  • the kit also contains instructions for carrying out the methods.
  • the method of the present invention is characterized by detecting the polymo ⁇ hisms provided herein, and is useful in gene diagnosis for detecting CYP3A4, CYP3A5 and/or GSTMl gene polymo ⁇ hisms. As long as the method is capable of detecting the aforementioned specific types of mutation which are clearly defined and characterized by the present invention, no limitation is imposed on the techniques to be employed in the method.
  • databases comprising sequence information pertaining to at least one the polymo ⁇ hisms of the present invention.
  • Software programs comprising use of a database of the present invention are also included.
  • said method further comprises digesting DNA encoding at least a part of the nucleic acid sequence containing the polymo ⁇ hism with a restriction enzyme that will cut, or will not cut, at or adjacent to one of the polymo ⁇ hic positions according to whether the polymo ⁇ hism is present.
  • a restriction enzyme that will cut, or will not cut, at or adjacent to one of the polymo ⁇ hic positions according to whether the polymo ⁇ hism is present.
  • Antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.
  • This example describes the identification of a variant of the known cytochrome P450 3A5 sequence (CYP4503A5).
  • PCR Polymerase chain reaction
  • PCR Polymerase chain reaction
  • the PCR amplification was performed in a total reaction volume of 50 microliters ( ⁇ l).
  • the final magnesium chloride concentration (2mM) was optimized empirically.
  • the final genomic DNA concentration was about 100 nanogram (ng) per reaction from 2 individuals.
  • the PCR reactions were performed using Perkin Elmer GeneAmp PCR kit (available from Perkin Elmer, Norwalk, CN) using Taq Gold DNA polymerase according to manufacturer's instructions and using the following primers.
  • a sequence comprising a polymo ⁇ hism identified from the sequencing is shown below the corresponding wildtype sequence, both the polymo ⁇ hic nucleotide and the corresponding wildtype nucleotide at the same position are shown in bold, and are listed below in Table 4.
  • Table 4 Newly Identified CYP4503A5 Gene Polymo ⁇ hism
  • SEQ ID NO:4 lists the sequence of the reference CYP4503A5 gene, includes a portion of the promoter and exon 1.
  • Example 2 This example describes the association of the Promoter -392 CYP4503A4 polymo ⁇ hism with survival rate of breast cancer patients receiving treatment with the chemotherapy drugs cyclophosphamide, cisplatin and l,3-bis-(chloroethyl)-l-nitrosourea.
  • Genomic DNA was isolated from peripheral blood lymphocytes of 86 chemotherapy naive female patients with metastatic or inflammatory breast cancer who participated in a trial for high-dose cyclophosphamide (CY), cisplatin and 1,3-bis- (chloroethyl)-l-nitrosourea (BCNU) chemotherapy with breast cancer using standard methods.
  • DNA from each patient was genotyped for the Promoter -392 CYP4503A4 single nucleotide polymo ⁇ hisms (SNPs) in the CYP3A4 gene.
  • Taqman assays were performed using DNA samples from each individual to identify the presence or absence of the Promoter -392 CYP4503A4 variant.
  • the primers identified in Table 5 were used:
  • PCR amplification was performed using IX Perkin-Elmer Taqman Reagent Mix #43C4447, about 900 nM 3A4-392 for primer, about 900 nM 3A4-392 rev primer, about 200 nM normal FAM-labeled probe having the nucleic acid sequence 5'- AGAGACAAGGGCAAGAGAGAGGCGAT-3' (SEQ ID NO: 19), and 200 nM variant TET-labeled probe having the nucleic acid sequence 5'-
  • GACAAGGGCAGGAGAGAGGCGA-3' (SEQ ID NO:20).
  • Thermal cycling was performed at an initial temperature of 50°C for 2 min followed by a denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 sec, primer annealing and extension was performed at 60°C 30 sec.
  • the fluorescence resulting from the release of labeled probe during PCR amplification and probe hybridization was measured using a fluorometer and the ratio of FAM to TET fluorescence was calculated to determine the occurrence of the polymo ⁇ hic site and homo- or heterozygosity compared to sequenced controls.
  • This example describes the association of the Promoter -147 CYP4503A5 polymo ⁇ hism described in Example 2 with survival rate of breast cancer patients receiving treatment with the chemotherapy drugs CY, cisplatin and BCNU.
  • the genomic DNA described above in Example 2 was genotyped for the Promoter
  • #43C444 7 with about 900 nM 3A5-147 for primer, about 900 nM 3A5-147 rev primer, about 150 nM FAM-labeled probe having the nucleic acid sequence 5'- CTGCAGCCCCACCTCCTTCTCC-3' (SEQ ID NO:23) and 250 nM variant NIC-labeled probe having the nucleic acid sequence 5'-CTGCAGCCCCGCCTCCTTCTC-3' (SEQ ID ⁇ O:24).
  • Thermal cycling was performed with at initial temperature of 50 °C for 2 min for activation of the Amperase UNG in the Taqman Reagent Mix followed by a denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 60°C for 30 sec, primer annealing and extension was performed at 60°C for 30sec.
  • the fluorescence resulting from the release of probe labels during PCR and probe hybridization was measured using a fluorometer and the ratio of FAM to VIC fluorescence was calculated to determine the occurrence of the polymo ⁇ hic site and homo- or heterozygosity compared to sequenced controls.
  • This example describes the association of the null GSTMl polymo ⁇ hism with survival rate of breast cancer patients receiving treatment with the chemotherapy drugs cyclophosphamide, cisplatin and BCNU
  • Example 2 The genomic DNA described above in Example 2 was genotyped for the null mutation of the GSTM 1 gene described in detail in Abdel-Rahman et al. , Cancer Letters, vol.
  • PCR amplification was performed at a MgCl 2 concentration of 2.5 mM in 1 X buffer

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Abstract

L'invention concerne des procédés de détection de gènes de variants présentant un polymorphisme associé à un métabolisme réduit d'un substrat sélectionné dans le groupe renfermant un substrat CYP3A4, un substrat CYP3A5 et un substrat GSTM1 chez un individu. Les procédés sont des procédés de génotypage permettant d'identifier des polymorphismes spécifiques dont on a découvert l'association avec un métabolisme réduit d'agents chimiothérapeutiques, tels qu'un cyclophosphamide et un BCNU. L'invention concerne également des nouvelles molécules d'acides nucléiques polymorphes utiles dans les procédés selon l'invention.
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Citations (3)

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WO1999013106A1 (fr) * 1997-09-10 1999-03-18 Axys Pharmaceuticals, Inc. Determination du genotype du gene humain cyp3a4
US6174684B1 (en) * 1999-08-11 2001-01-16 Trustees Of The University Of Pennsylvania CYP3A4 NFSE variant and methods of use therefore
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WO1999013106A1 (fr) * 1997-09-10 1999-03-18 Axys Pharmaceuticals, Inc. Determination du genotype du gene humain cyp3a4
US6183963B1 (en) * 1998-10-23 2001-02-06 Signalgene Detection of CYP1A1, CYP3A4, CYP2D6 and NAT2 variants by PCR-allele-specific oligonucleotide (ASO) assay
US6174684B1 (en) * 1999-08-11 2001-01-16 Trustees Of The University Of Pennsylvania CYP3A4 NFSE variant and methods of use therefore

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FELIX ET AL.: "Association of CYP3A4 genotype with treatment-related leukemia", PROC. NATL. ACAD. SCI. USA, vol. 95, October 1998 (1998-10-01), pages 13176 - 13181, XP001019043 *
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