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WO2001032928A2 - Methodes permettant de determiner une hypersensibilite a un agent - Google Patents

Methodes permettant de determiner une hypersensibilite a un agent Download PDF

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Publication number
WO2001032928A2
WO2001032928A2 PCT/US2000/030474 US0030474W WO0132928A2 WO 2001032928 A2 WO2001032928 A2 WO 2001032928A2 US 0030474 W US0030474 W US 0030474W WO 0132928 A2 WO0132928 A2 WO 0132928A2
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WO
WIPO (PCT)
Prior art keywords
genes
hypersensitivity
gene expression
agent
cells
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Application number
PCT/US2000/030474
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English (en)
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WO2001032928A3 (fr
WO2001032928A9 (fr
Inventor
Spencer Farr
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Phase-1 Molecular Toxicology
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Priority to AU14660/01A priority Critical patent/AU1466001A/en
Publication of WO2001032928A2 publication Critical patent/WO2001032928A2/fr
Publication of WO2001032928A9 publication Critical patent/WO2001032928A9/fr
Publication of WO2001032928A3 publication Critical patent/WO2001032928A3/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the invention generally relates to methods, compositions and devices for identifying individuals who are hypersensitive to a given agent.
  • clozapine is a very effective drag for treating moderate to severe depression and with the majority of patients shows no toxic side effects at the recommended doses. Yet at the same dose (usually 300 mg), approximately 1% of the patient population develop agranulocytosis, a severe blood disorder.
  • T A T"l_ fialuridine, carbamazepine, Trovan (frovafloxacin), Seldane (terfenadine), hismanol, dihydrolazine, warfarin, phenytoin, omeprazole, diazepam, haloperidol, perphenazine, perhexiline, phenformin, tolbumamide, penicillin, clozapine, aminopurine, quinidine and remoxipide.
  • Hypersensitive individuals are discovered the hard way; they exhibit toxic side effects that most people do not.
  • the mechanisms of toxicity are specific and usually different for each drug or compound, the hypersensitive populations are also different and specific for each drag or compound.
  • the invention relates to a method of identifying hypersensitivity in a subject by obtaining the gene expression profile of specific genes associated with hypersensitivity of the subject suspected to be hypersensitive and identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity.
  • the gene expression profile of the subject may be compared with the gene expression profile of individuals who have an acceptable response and compared with other hypersensitive individuals.
  • the embodiment also includes, for example, identifying hypersensitivity to an agent in a subject, where the agent may be a pharmaceutical agent, industrial, household or other chemical or compound. Exemplary pharmaceutical agents are disclosed in Table 1.
  • the gene expression profile of the subject that is obtained may comprise a profile of levels of mRNA or cDNA.
  • the gene expression profile may be obtained by using an anay of nucleic acid probes complementary to the genes associated with hypersensitivity.
  • the genes used may comprise at least two genes, at least 3, 4, 6, 7, 8, or 9 genes predetermined to be associated with hypersensitivity, and may also comprise at least 5, at least 10, at least 25, at least 50, at least 100, at least 250 or more genes determined to be associated with hypersensitivity.
  • Genes associated with hypersensitivity and used in this invention may, for example, comprise genes from a variety of different cell types, including, but not limited to, genes from multiple types of tissues, organs or systems or genes from a single type of tissue, organ or system.
  • organs and tissues include the liver, kidneys, heart, brain, thyroid, lung, pancreas, muscle, brain, testes, ovaries, spleen, stomach, intestines, colon, rectum, eyes, muscle, skin, and bone.
  • Exemplary types of cells include liver cells such as, Kupfer cells, sinusoidal cells, ito cells, hepatocytes, bile duct epithelial cells, hepatic venule endothelial cells and sinusoidal epithelial cells.
  • a further embodiment encompasses the expression profile of the genes predetermined to be associated with hypersensitivity where expression of the genes is related to prevention or repair of toxic damage at the nucleotide, protein, macromolecule, organelle, cell, tissue, organ or system level.
  • the gene expression profile may comprise a profile of protein expression levels, where the proteins are encoded by genes associated with hypersensitivity. The level of expression of the proteins may be directly related to the prevention or repair of toxic damage at the protein, nucleotide, macromolecule, organelle, cell, tissue, organ or system level.
  • An additional embodiment includes protein expression profiles, where the proteins are encoded by genes associated with hypersensitivity, and the expression of the genes is, for example, associated with response to the presence of an agent, such as a toxic agent. Exemplary agents that can induce a characteristic profile of protein expression associated with hypersensitivity include those agents listed in Table 1.
  • the gene expression profile may be obtained from a sample from the subject, which sample may be from a cell or tissue sample and may comprise cells of different cell types.
  • the sample may comprise, for example, white blood cells, skin, spinal fluid or organ biopsy material.
  • the sample may comprise, for example, blood, tissue, urine, spinal fluid or serum.
  • cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. These primary cell cultures are then exposed to the agent.
  • Co-cultures are also encompassed in the invention and are grown from two or more cell types that reflect, for example, the cell types involved in systemic toxicity. These co-cultures would then be exposed to the agent of interest.
  • the gene expression profiles of samples from normal individuals, hypersensitive individuals or cell cultures are determined for individual agents using the methods herein described to determine drag-drug interactions.
  • the gene expression profiles are compared to determine whether the multiple agents, for example two or more agents, elicit the same or similar gene expression profiles in the samples.
  • the expression of the same or similar pattem(s) of toxic response genes for two or more compounds in either normal or hypersensitive individuals is indicative that a drag-drag interaction, also described as a synergistic toxic effect, can be present if the agents are administered together, for example, during the same time period or in the same dose.
  • genes used in the gene expression profile may include, but are not limited to, genes, and the proteins which they encode, which are associated with toxic outcomes affecting the pulmonary system, cardiovascular system, nervous system, digestive system, immune system, reproductive system, endocrine system, vision or skin.
  • exemplary types of toxicity include cardiotoxicity, blood toxicity, liver (hepatic) toxicity, kidney (renal) toxicity, neural toxicity, skin toxicity, immunotoxicity, and pulmonary toxicity.
  • Exemplary genes associated with specific organ or system toxic outcomes are disclosed in Table 5.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, altered lipid metabolism, altered thyroid function, organ hypertrophy, skin initation, skin sensitization, tumor formation, dementia, inflammation, myelosuppression, peripheral neuropathy, necrosis, signal refractivity, spreading, transformation, retinopathy or optic atrophy.
  • genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the digestive system or the organs and tissues which comprise the digestive system, for example, the liver, kidneys, colon, bladder, pancreas, stomach, intestines, rectum, or gallbladder.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with exemplary toxic outcomes such as, but not limited to, proteinuria, glomerulitis, nephritis, renal damage, renal failure, liver weight change, cholestasis, pancreatitis, liver steatosis, hyperplasia, fatty liver, jaundice, hepatitis, mutagenesis, or altered bile flow.
  • exemplary toxic outcomes such as, but not limited to, proteinuria, glomerulitis, nephritis, renal damage, renal failure, liver weight change, cholestasis, pancreatitis, liver steatosis, hyperplasia, fatty liver, jaundice, hepatitis, mutagenesis, or altered bile flow.
  • genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the pulmonary system or the organs and tissues which comprise the pulmonary system, for example the lungs or trachea.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, lung fibrosis, pulmonary edema or lung airway reactivity.
  • the genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the cardiovascular and circulatory systems or the organs, fluids and tissues which comprise the cardiovascular and circulatory systems, for example, the heart, spleen, arteries, blood vessels, blood or blood cells, including genes associated with toxic outcomes associated with bone manow.
  • the genes used in the gene expression profile include those genes, and the proteins which they encode, associated with exemplary toxic outcomes such as, but not limited to, tachycardia, anhythmia, leukemia, neutropenia, hematological alteration, hypotension, hypertension or agranulocytosis.
  • the genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the nervous system or the organs and tissues which comprise the nervous system, for example, the brain, spinal cord or nerves.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, neurodegeneration or neurotoxicity.
  • genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the immune system or the organs and tissues which comprise the immune system, for example, the thymus, lymph nodes or lymph glands.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, a change in thymic weight or immunosuppression.
  • genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the reproductive system or the organs and tissues which comprise the reproductive system, for example the testes, ovaries, fallopian tubes or uterus.
  • genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, teratogenesis, loss of fertility, alteration in sperm count, alteration in testes weight or alteration in testosterone levels.
  • the genes used in the gene expression profile include those genes, and the proteins which they encode, associated with cellular manifestations of toxicity such as, but not limited to, apoptosis, cell adhesion, autophagocytosis, cell division, chemotaxis, cell cycle anest, circadian rhythm, cytokine release, differentiation, de-differentiation, mitochondrial damage, migration, mutation, oncosis, recombination, senescence, peroxisome proliferation, polyploidy, signal refractivity, spreading, transformation or necrosis.
  • genes involved, and the proteins which they encode may also include those associated with a specific ethnic group, sex or age group.
  • genes or proteins used in the expression profile may also include the genes, and the proteins or amino acids which they encode, which are selected from the genes disclosed in (or genes comprising sequences disclosed in) Table 3, Table 4, Table 5, Table 6, Table 8, Table 10 and Table 11.
  • the method includes obtaining a gene expression profile of genes comprising different cell types, of the subject, determining if the gene expression profile of the subject comprises a pattern of gene expression associated with hypersensitivity to an agent, and withholding that agent from those subjects who are hypersensitive or altering the therapy and closely monitoring the subjects who are hypersensitive for toxic effects.
  • a method of identifying a plurality of genes associated with hypersensitivity to an agent comprising comparing the gene expression profile of cells treated with an agent with the gene expression profile of cells not treated with the agent and identifying genes that have altered expression due to exposure to the agent in the treated cells.
  • the cells may comprise, for example, a number of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent.
  • the cells may also comprise cells from of different cell types where all the cell types are derived from a single type of tissue, organ or system.
  • the organs or tissues from which cell types may be derived include, but are not limited to, the kidneys, liver, lungs, heart, brain, spleen, thyroid, bone, muscle, intestine, stomach, pancreas, testes, ovaries, colon or skin.
  • the invention also relates to a method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent by comparing the gene expression profile of one or more cell types, for example, at least 2, at least 3, at least
  • the method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent comprises comparing the gene expression profile of one or more cell types, for example, at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100 or at least 250, of a subject known to be hypersensitive to the agent before treatment with the agent with the gene expression profile of the one or more cell types of the subject after treatment with the agent and identifying genes from the cell types having a pattern of differential gene expression associated with hypersensitivity to the agent.
  • the method of identifying proteins having a pattern of differential protein expression indicative of hypersensitivity to an agent comprises comparing the protein expression profile of one or more cell types ofa subject known to be hypersensitive to the agent before treatment with the agent with the protein expression profile of the one or more cell types of the individual after treatment with the agent and identifying proteins from the cell types having a pattern of differential protein expression associated with hypersensitivity to the agent.
  • an anay for the identification of a gene expression profile indicative of a hypersensitivity to an agent which comprises gene probes, for example, nucleic acid sequences which comprise a gene sequence associated with hypersensitivity to the agent, associated with the hypersensitivity to the agent.
  • the genes are selected from the genes identified by methods disclosed herein or are selected from those genes disclosed in whole or in part in Table 3, Table 4, Table 5, and Tables 6, 8, 10 and 11.
  • the anay comprises for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 250 different gene probes.
  • Exemplary anays include, for example, gene probes supported on glass slides or nylon membranes with fluorescent or radio labels, amplified fragment length polymorphism (AFLP) methods or Northern Blots.
  • AFLP amplified fragment length polymorphism
  • the invention further encompasses a database of genes associated with hypersensitivity to an agent.
  • the genes are those identified by methods disclosed herein or are selected from those genes disclosed in whole or in part in Table 3 and Table 4, Table 5 and Tables 6, 8, 10 and 11.
  • the database of genes may comprise, for example, genes associated with altered lipid metabolism, cholestasis, immunosuppression, pancreatitis, agranulocytosis, tumor formation, teratogenesis, liver steatosis, apoptosis, cell adhesion, autophagocytosis, cell cycle anest, circadian rhythm, cytokine release, differentiation, migration, oncosis, recombination, senescence, signal refractivity, spreading, transformation, peroxisome proliferation, necrosis, glomerulitis, nephritis, anhythmia, hypotension, hypertension, leukemia, neutropenia renal damage, renal failure, pulmonary edema, neurotoxicity or retinopathy.
  • An additional embodiment includes an apparatus for identifying hypersensitivity in a subject comprising means for obtaining a gene expression profile of a number of genes associated with hypersensitivity of the subject suspected to be hypersensitive; and means for identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity, thereby to identify hypersensitivity in the subject.
  • the pattern of expression may be detected in a cell, such as an immune cell, such as a leukocyte, e.g. a lymphocyte.
  • devices for detection of gene expression profiles comprising nucleic acid sequences for detecting expression of the nucleic acids disclosed in the Tables, for example by hybridization.
  • Such devices include, for example, immobilized nucleic acid anays.
  • Figure 1 is a graph illustrating gene expression changes associated with toxicity caused by sfreptozotocin.
  • Figure 2 is a graph illustrating co-induction of genes for hepatocyte growth factor receptor and glutathione transferase.
  • Figure 3 is a graph illustrating a portion ofa gene expression profile from heart muscle tissue after exposure to the cardiotoxin, doxombicin.
  • Figure 4 is a graph illustrating a portion ofa gene expression profile from liver tissue after exposure to the peroxisome proliferation caused by WY 14,643.
  • Figure 5 is a graph illustrating a portion ofa gene expression profile from liver tissue after exposure to the anti-neoplastic compound, carbamazapine.
  • Figure 6 is a chart illustrating the result of testing for penicillin hypersensitivity amongst a group of penicillin sensitive and penicillin refractive individuals by using a 180 gene penicillin anay.
  • Figure 7 is a chart illustrating the result testing for penicillin hypersensitivity amongst a group of penicillin sensitive and penicillin refractive individuals by using a 20 gene penicillin anay.
  • Figure 8 is a chart illustrating 20 discriminator genes analyzed for co-regulation.
  • Figure 9 is a graph illustrating the results of a Taqman® assay in a penicillin sensitive person.
  • Figure 10 is a graph illustrating the results of a Taqman® assay in a penicillin refractive person.
  • Table 1 is a list of pharmaceutical agents which potentially can cause greatly heightened toxic responses in some individuals.
  • Table 2 is a list of industrial agents which potentially can cause greatly heightened toxic responses in some individuals.
  • Table 3 is a list of genes, altered expression patterns of which can indicate and render an individual hypersensitive to drugs and chemical agents.
  • Table 4 is a list of genes, altered expression patterns of which can indicate and render an individual hypersensitive to drags and chemical agents.
  • Table 5 is a list of genes associated with specific manifestations of organ or system toxicity.
  • Table 6 is a list of genes that can be associated with specific cellular manifestations of toxicity.
  • Table 7 lists compounds for which gene expression data in either human cells, rats or both has been generated.
  • Table 8 lists genes whose expression was measured when rats were exposed to the cardiotoxin doxombicin.
  • Table 9 lists cell types in organs of toxicity.
  • Table 10 lists the characterization of genes which were isolated and sequenced from gel bands.
  • Table 11 lists the genes that are useful discriminator genes.
  • hypersensitivity in a subject is determined by obtaining from the subject a sample from which can be determined the gene expression profile of genes associated with hypersensitivity, and identifying in the gene expression profile the presence or absence of a pattern of gene expression of the genes associated with hypersensitivity, thereby to identify hypersensitivity in the individual.
  • the terms 'gene', 'polynucleotide', 'nucleotide' and 'nucleic acid' are interchangeable and refer to polynucleotide sequences, which for example, encode protein products and encompass mRNA, cDNA, single stranded DNA, double stranded DNA and fragments thereof.
  • the terms "protein”, “polypeptide”, and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids as well as other modifications known in the art.
  • a toxic response gene can be defined as a gene whose message or protein level is altered by adverse stimuli.
  • the specific set of genes that cells induce is dependent upon the type of damage or toxic threat caused by the agent and which organs are most threatened.
  • genes which encode functions not appropriate under conditions of toxic injury may be down-regulated.
  • 'toxic outcome' refers to the microscopic or macroscopic symptoms, physiological, morphological or pathological changes which are observed as a result of exposure to an agent.
  • a 'toxic response' as used herein refers to a cellular, tissue, organ or system level response to exposure to an agent and includes, but is not limited to, the differential expression of genes and/or proteins encompassing both the up- and down-regulation of such genes; the up- or down-regulation of genes which encode proteins associated with the repair or regulation of cell damage; or the regulation of genes which respond to the presence of an agent.
  • a 'gene expression profile associated with hypersensitivity' refers to the pattern of relative levels of gene expression found to be associated with hypersensitivity. Gene expression profiles may be measured in a sample, such as samples comprising a variety of cell types and may, for example, comprise blood, urine, spinal fluid or serum.
  • a 'protein expression profile associated with hypersensitivity' is defined as the pattern of relative levels of protein expression where said proteins are encoded by genes determined to be associated with hypersensitivity. For each gene expression profile that is determined, a conesponding 'protein expression profile associated with hypersensitivity' may be determined.
  • 'up-regulation' and 'induction' are used interchangeably herein and refer to the regulation of gene expression, specifically the turning on of a particular gene(s).
  • 'down-regulation' and 'repression' are used interchangeably herein and refer to the suppression of expression of a particular gene(s).
  • An 'agent' to which an individual is hypersensitive is defined as any substance to which an individual may be hypersensitive and includes, but is not limited to, drags, household chemicals, industrial chemicals and other chemicals and compounds to which individuals may be exposed.
  • 'Hypersensitivity' refers to the exaggerated micro- or macroscopic responses of cells, tissues, organs or systems to low or average doses of an agent. These responses may lead to observable symptoms such as dizziness or nausea and can also result in toxic outcomes. Hypersensitivity often results in toxic side effects that are different, in either degree or kind, from the response of the majority of patients at the recommended dose.
  • Hypersensitivity may be characterized by, but is not limited to, the differential expression of genes when compared to the response ofa similar individual who is not hypersensitive to a given agent. Hypersensitive individuals do not have normal gene expression patterns of key toxicologically relevant genes either prior to, or after, exposure to an agent.
  • differential gene expression refers to the change in expression levels of genes, and/or proteins encoded by said genes, in cells, tissues, organs or systems upon exposure to an agent.
  • differential gene expression includes differential franscription and translation, as well as message stabilization. Differential gene expression encompasses both up- and down-regulation of gene expression.
  • the term 'individual' is used interchangeably with the term 'subject' and 'patient' and refers to a mammal, preferably the primate, more preferably the human.
  • the term 'normal individual' or 'normal subject' refers to individuals who exhibit the same or similar dose response curves to an agent as does the majority of the exposed population. Most drags at high enough dosages will cause a toxic response, therefore a 'normal toxic response' refers to the toxic response elicited in an average or normal individual at high doses of an agent.
  • the term 'sample' as used herein refers to samples for testing or analysis.
  • the samples may comprise cells or tissue samples and may be, for example, blood, urine or serum.
  • Samples are characterized in a prefened embodiment by comprising at least two different genes and may also include genes from multiple cell types. Samples include, but are not limited to, those of eukaryotic, mammalian or human origin. As used herein, “anay” and “microanay” are interchangeable and refer to an anangement of a collection of nucleotide sequences in a centralized location. Anays can be on a solid substrate, such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any permutations thereof.
  • the nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences.
  • "Penicillin sensitive” refers to individuals who exhibit hypersensitivity to penicillin, for example, a higher than average immune response to penicillin.
  • the immune response can be a hypersensitive response of any type, for example Type I, II, III, or IV. Hypersensitive reactions can include but are not limited to anaphylaxis, skin rash, and hives. Hypersensitive responses also include hypertoxicity.
  • "Penicillin refractive" or "penicillin insensitive” or “penicillin non-sensitive” as used herein refers to individuals who exhibit a normal or non-hypersensitive response to penicillin.
  • PBL peripheral blood lymphocytes
  • total RNA is isolated from tissue samples using the following materials: Qiagen RNeasy midi kits, 2-mercaptoethanol, liquid N 2 , tissue homogenizer, dry ice. It is important to take precautions to minimize the risk of RNA degradation by RNases by wearing gloves at all times and to inhibit RNase activity in work areas and equipment by treating with an RNase inhibitor such as with "RNase Zap" (Ambion® Products, Austin, TX). Autoclaving tips and microfiige tubes does not necessarily eliminate RNase enzymes and its RNA degradation activities. Samples are kept on ice when specified . Protocol which can be used is based on Qiagen® RNeasy® midi kit.
  • RNA isolation technique is used for RNA isolation from human PBL and can be modified readily by one of skill in the art to accommodate different amount of human PBLs.
  • the human PBL is preferably taken from circulating blood of a human donor. However, human PBL can also be obtained from lymph nodes, spleen, and other tissues into which human PBLs circulate. If tissue containing human PBL is used, then the tissue needs to be microdissected. One way is to physically break the tissue by placing it on a double layer of aluminum foil which is then placed within a weigh boat containing a small amount of liquid nifrogen. The aluminum foil is folded around the tissue and then the tissue is stmck by a small foil- wrapped hammer to administer mechanical stress forces.
  • RNA is kept on dry ice when other samples are being weighed.
  • a buffer is added to the sample to aid in the homogenization process.
  • An example of a buffer which can be used is RLT (Qiagen®) buffer.
  • the tissue is homogenized using any type of commercially available homogenizer (i.e. IKA Ultra Tunax T25 homogenizer, Virtishear Cyclone 750W rotor/stator homogenizer (Virtis item # 278077, etc.) can be used with the 7 mm microfine sawtooth shaft and generator (195 mm long with a processing range of 0.25 ml to 20 ml, item # 372718).
  • DNA or RNA is isolated from human PBLs obtained from a human donor.
  • lymphocytes can be isolated from blood by separating the blood over a gradient, for example a sucrose gradient or PercollTM or FicollTM gradient. Lymphocytes can be distinguished from non-lymphocyte contaminates by morphology, size and scatter by flow cytometry, or by cell surface markers such as CD2, CD3, CD4, or
  • lymphocytes which are cultured in vitro are non-adherent but in some instances, lymphocytes can be adherent or non-adherent depending on several factors, for example, activation state of lymphocytes, receptors expressed on lymphocytes, and culture media contents.
  • adherent cells are more problematic than non-adherent cells because of the necessity of an extra step to separate the adherent cells from the tissue culture container.
  • a skilled artisan may solve this problem by treating the cells with cold PBS/EDTA solutions or an equivalent and use any number of commercially available kits, for example, from Qiagen or Ambion, to isolate the DNA or RNA from the cells.
  • total RNA of high quality and high purity can be isolated from cultured cells by using Qiagen RNeasy midi kits and 2-mercaptoethanol. This embodiment is exemplified in Example 2 infra. Precautions should be taken to minimize the risk of RNA degradation by RNases by wearing gloves, treating work areas and equipment with an
  • RNase inhibitor for example RNase Zap (Ambion® Products, Austin, TX), and keeping samples on ice.
  • this total RNA isolation technique can be used for any type of cell, including but not limited to human lymphocytes and cell derived from particular organs such as kidney, liver, lung, breast, neuronal cells, skin, intestine, such as HepG2, Caco-2, MCF-7, Jurkat, Daudi, HL-60, MCL-5, SKBr-3,
  • cells are checked under the microscope to confirm viability.
  • Cells are then dosed with an agent, which can be a drag, chemical, or pharmaceutical composition, when they reach confluence.
  • an agent which can be a drag, chemical, or pharmaceutical composition
  • the cells are at least about 20% confluent, more preferably at least about 40% confluent, even more preferably at least about 60% confluent, and even more preferably about 80% confluent. It is preferable to avoid isolating RNA from flasks that have reached 100% confluence because the cells are no longer growing in log phase.
  • the adherent cells are washed and freshly prepared buffer, for example RLT buffer (RLT buffer requires the addition of 10 ⁇ l beta mercaptoethanol for each 1.0 ml RLT), is added directly to the cell culture flask.
  • RLT buffer requires the addition of 10 ⁇ l beta mercaptoethanol for each 1.0 ml RLT
  • the amount of RLT buffer differs with tissue container size. Enough RLT buffer is added to cover the surface area in which the adherent cells are growing such that most of the adherent cells come into contact with the RLT buffer.
  • T-75 flasks receive about 3 ml RLT buffer and T-175 flasks receive about 5 ml RLT buffer. It is preferable to lightly agitate the flasks at this point. Cells exposed to RLT buffer become a gelatinous layer.
  • the cells are allowed to sit for 4 minutes, then fluid is withdrawn and is placed into and RNase-free tube. An equivalent volume of 70% ethanol is added to each tube and vortexed to distribute evenly. In the event that a precipitate with a string-like appearance forms, it is acceptable to remove and discard this string-like precipitate.
  • the fluid is applied to a spin column, centrifuged, and the column is washed and subsequently eluted for RNA samples.
  • the elution can be precipitated using the LiCl precipitation protocol and resuspended in RNA storage buffer for future storage.
  • the yield can be between 200-400 ⁇ g of total RNA from a T-75 flask with greater than 50% confluency.
  • the isolated DNA or RNA is amplified to generate a product which can be attached to a subsfrate.
  • the substrate is a solid subsfrate (i.e. glass slide).
  • the amplification process involves using primers which have a reactive group (i.e. amine group or derivative thereof) on one end of the primer, which is incorporated into the amplification product.
  • a reactive group i.e. amine group or derivative thereof
  • reactive primers i.e. amine group or derivative thereof
  • Amine Primers from Synthegen.
  • the gene fragments which are attached to the glass slide can vary in length. The more nucleotides ofa gene that are in the anay, the tighter the binding and the greater the specificity in binding can occur.
  • the desired length ofa gene or a fragment thereof that is to be included in the anay should take into consideration the balance between a high specificity of binding obtained with a long (i.e. >1 kb) gene sequence with the high mutational rate associated with a longer fragment.
  • the gene fragments attached to the glass slide are at least about 50 base pairs (bp) in length, more preferably at least about 100 bp in length, more preferably at least about 200 bp, even more preferably at least about 300 bp, even more preferably at least about 400 bp, even more preferably at least about 500 bp in length.
  • the gene fragments are about 500 bp in length.
  • the region of a gene that is used to attach to a solid substrate to generate an anay can be any portion of the gene, coding, non-coding, 5' end, 3' end, etc.
  • about 500 base pairs of the 3' end of canine gene related to toxicological responses are selected to be included in an anay.
  • a method is to attach an amine group, a derivative of an amine group, another group with a positive charge or another group which is reactive to one end of a primer that is used to amplify a gene or a gene fragment to be included in the anay. Subsequent amplification of a PCR product will then inco ⁇ orate this reactive group onto one end of the product.
  • the amplified product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified PCR product and become covalently attached to the glass slide.
  • a solid substrate such as a glass slide
  • fluorescence-labeled single strand (or "first strand") cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microanay slides spotted with DNA specific for toxicologically relevant genes. This is exemplified in Example 8-14.
  • the materials needed to practice this embodiment are: total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice.
  • the CyTM dyes may be obtained from Amersham.
  • the embodiment may also be practiced with equivalents of the materials listed above, for example, Superscript II may be replaced with an equivalent enzyme and Cy5 and Cy3 may be replaced with another fluorescent dye.
  • RNA for example 20 ⁇ g of total RNA or 2 ⁇ g of mRNA
  • the volume is no more than 14 ⁇ l. If RNA is too dilute, the samples are concentrated to a volume of less than 14 ⁇ l in a centrifuge with vacuum (i.e. Speedvac) without heat.
  • the Speedvac should be capable of generating a vacuum of 0 Milli-Ton so that samples can freeze dry under these conditions. It is preferable for the tubes containing RNA to be kept on ice to avoid RNA degradation until the next step is ready to proceed.
  • cDNA samples are amplified from RNA templates.
  • a mixture of fluorescent dyes is made for labeling the cDNA samples.
  • a variety of dyes can be used.
  • Cy3 dye which is pink-red
  • Cy5 dye which is blue
  • the Cy dyes are light sensitive, therefore, any solutions or samples containing Cy-dyes should be kept out of light, i.e. cover with foil.
  • Example 9-12 discloses prefened mixtures and methods of using Cy3 and Cy5 dyes for labeling cDNA samples and purification steps therewith.
  • Cy3 dye mixture is incubated with the cDNA of each treated sample and Cy5 dye mixture is incubated with the cDNA of each confrol sample.
  • Cy5 dye mixture is incubated with the cDNA of each confrol sample.
  • a visible pellet can be seen which is pink/red for cDNA incubated with Cy3 and blue for cDNA incubated with Cy5. It is recommended that the tubes are centrifuged at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the cDNA sample (or cDNA probe) is seen spread on one side of the tube instead of a tight pellet. If the pellet is white (no pink/red or blue), it is likely that the reaction has not occuned to maximal efficiency. Purification of fluorescent probes
  • probes are purified by binding to a resin.
  • the binding resin can be obtained by itself or from a kit provided by any number of commercial sources, i.e. Qiagen, Promega, etc.
  • Incorporation of fluorescence into cDNA probes can be achieved by using a number of methods.
  • the following material is used: 384 well, 100 ⁇ l assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent).
  • cDNA probes Prior to use as a cDNA probe in hybridization, cDNA probes are purified and concentrated as exemplified in Example 10.
  • Cy3 and Cy-5 fluorescence are analyzed using a fluorimeter, luminometer, flow cytometer, or any equivalent device which can detect different fluorescent dyes at different wavelengths.
  • the Wallac 1420 workstation programmed for reading Cy3-Cy-5 is used.
  • a typical range for Cy-3 (20 ⁇ g) is 250-700,000 fluorescence units.
  • a typical range for Cy-5 (20 ⁇ g) is 100-250,000 fluorescence units.
  • Prefened settings for the Wallac 1420 fluorescence analyzer are as follows: Cy3
  • Hybridization of labeled cDNA probes to single stranded, covalently bound DNA target genes on glass slide microa ⁇ ays can be accomplished by a variety of methods.
  • the following material are used: formamide,
  • Hybridization buffer is prepared with consideration towards stringency. Stringency can be varied by increasing or decreasing the amount of SSC and detergent (i.e. SDS, Triton, etc.). Stringency can also be varied by the temperature at which the hybridization occurs. A higher temperature tends towards high stringency conditions. A skilled artisan can determine, in a stepwise fashion, the stringency of the hybridization buffer desired. Clean slides and coverslips are desirable and can be obtained using N 2 stream.
  • Hybridization buffer is added to dried probe and mixed in the dark at room temperature and then brought to a higher temperature in a heat block. Each probe can remain in a heat block until it is ready for hybridization.
  • the probe is applied to a slide or to a coverslip and then covered with slide. It is highly preferable to avoid the material at the bottom of the tube and to avoid generating air bubbles. This may mean leaving some residual volume in the pipette tip. Slides are then placed in a hybridization chamber, wrapped to prevent the liquids from desiccating.
  • One problem that can occur with overly dried slides is increased fluorescence on the edge of the spot containing the target gene fragment to which the labeled cDNA probe binds.
  • the hybridization chamber can have a built-in humidity gauge to avoid desiccation of the slides.
  • the slides are placed in a 42°C humidity chamber in a 42°C incubator for 18 to 24 hours. It is preferable to avoid probes or slides sitting at room temperature for long periods.
  • all non-specifically bound cDNA probe should be removed from the anay.
  • removal of all non- specifically bound cDNA probe can be accomplished by washing the anay using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. Equivalents of SSC and SDS may also be used as substitutes. It is highly preferable that great caution be used with the standard wash conditions since deviations can affect data significantly.
  • glass buffer chambers and glass slide holders are filled with heated SSC buffer with sufficient volume to submerge the microanays. It is important to exercise caution in heating of the SSC buffer since a high temperature may strip off the probes, preferably the temperature is at most about 60°C, more preferably at most about 50°C, even more preferably at most about 40°C, and even more preferably at most about 35°C. A skilled artisan can vary the concentration of SSC in the buffer according to the stringency desired.
  • the slides are placed in buffer which may contain SSC and/or detergent (i.e. SDS, Triton, etc.) and the coverslips are dislodged and fall off the slide within several minutes of submersion.
  • very gentle agitation may be administered to the chamber in which the wash is being conducted to dislodge the coverslips.
  • the slides with the hybridized probes are subjected to several rounds of washes with different conditions.
  • a detergent i.e. SDS
  • SDS a detergent
  • the slides are washed in this buffer before a final wash in nanopure water.
  • the slides are dried in a manner that will minimize background signal of the array.
  • a prefened method of drying is to use a folded paper towel underneath the slide and a gently dabbing motion on the slide with a tissue. It is important that the slides do not air dry since this will lead to increased background.
  • the pattern of gene expression characteristic of hypersensitivity is predetermined, and is, for example, provided in a database.
  • the hypersensitivity of the subject can be conveniently and rapidly determined.
  • the invention provides a large number of predetermined gene expression patterns of genes associated with hypersensitivity, for example in a database, so that a large number of genes can be rapidly analyzed and compared in the subject. Analysis of information about expression of a wide spectrum of genes associated with hypersensitivity facilitates the rapid determination of hypersensitivity of a subject to an agent, or multiple agents.
  • the differential gene expression profile associated with a given agent can be determined for a given agent using, for instance, eukaryotic or mammalian cells or cell lines or animal models and exposing a population of the eukaryotic or mammalian cells or cell lines or animal models to an agent and comparing their gene expression to the same type of eukaryotic or mammalian cells or cell lines or animal models from an untreated population to determine the gene expression profile associated with hypersensitivity.
  • Hypersensitivity to an agent for example, a pharmaceutical drag or household, industrial or other chemical, can be rapidly determined with samples from an individual or group of individuals by treating the sample(s) with an agent and comparing the gene expression profile with the gene expression profile associated with hypersensitivity determined previously for a particular agent and, for instance, stored in a database and accessed and compared with associated software.
  • Table 1 lists approximately 200 drags sold in the U.S. and Europe. There are individuals who are hypersensitive to the toxic side effects of each of these drags. Table 2 lists at least 100 major industrial chemicals for which there is documented evidence of toxicity due to occupational exposure. For each of these chemicals there are individuals whose toxic response is heightened compared to the majority of the population.
  • multiple genes are analyzed.
  • the number of genes, associated with hypersensitivity, whose expression levels are determined and which comprise the gene expression profile is large; for example, one or more, at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, or at least 250.
  • the present invention also encompasses gene expression profiles where the number of genes is greater than 400, 500, 600 or more.
  • the genes, whose expression levels comprise the gene expression profile are drawn from a variety of cell types.
  • the genes whose expression levels comprise the gene expression profile, are drawn from cells of a number of different tissues or organs.
  • cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. These primary cell cultures are then exposed to the agent. Cell cultures established from the appropriate tissues of hypersensitive individuals are more sensitive to the toxic effects of the agent than cultures established from normal individuals. This hypersensitivity is reflected in the gene expression patterns elicited from the cell cultures.
  • cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. Co-cultures would be grown from two or more cell types that reflect the cell types involved in systemic toxicity. These co-cultures are then exposed to the agent of interest. Cell co-cultures established from the appropriate tissues of hypersensitive individuals are more sensitive to the toxic effects of the compound than co-cultures established from normal individuals. This hypersensitivity is reflected in the gene expression patterns elicited from the cell co-cultures.
  • the gene expression profile consisting of the expression levels of multiple genes includes genes drawn from a single cell, tissue or organ type, and the profile is examined to determine the association of the gene expression profile with hypersensitivity.
  • the relative expression levels of two or more genes in the gene expression profile associated with hypersensitivity can be determined and can be relevant to a determination of hypersensitivity.
  • Hypersensitive individuals will have profiles of expression of relevant toxicity genes that are distinct from individuals who are not hypersensitive.
  • gene expression profiles from normal individuals, hypersensitive individuals or cell cultures are established for individual agents to determine possible toxic drag-drag interactions when patients (normal or hypersensitive individuals) are treated with multiple drags.
  • the expression of the same pattern of toxic response genes for two or more compounds in either normal or hypersensitive individuals indicates that the two or more compounds, taken together, will often show a synergistic toxic effect.
  • Gene expression profiles for each compound, determined in vitro or in vivo allows prediction of the severe toxicity if the two compounds were taken together.
  • the gene expression profile of genes associated with certain disease states is analyzed. Normal individuals can become temporarily hypersensitive to the toxicity of certain drags because of disease states. Hypersensitivity is present in normal individuals when toxic defense mechanisms are temporarily compromised. For example, an individual who suffers from AIDS-induced immunosuppression will be hypersensitive to the toxic effects of immunosuppressive compounds such as cyclosporin A. An individual suffering from pulmonary edema due to viral infection will be temporarily hypersensitive to compounds such as bleomycin which elicit pulmonary edema as a toxic side-effect.
  • the method includes obtaining a protein expression profile of a number of proteins encoded by genes of the subject, determining if the protein expression profile of the subject comprises a pattern of protein expression associated with hypersensitivity to an agent, and withholding the agent from those individuals or altering the therapy or dosage and closely monitoring the individual for toxic effects.
  • a method of identifying a number of genes associated with hypersensitivity to an agent comprising comparing the protein expression profile, where the proteins are encoded by the genes identified as associated with hypersensitivity to the agent, of cells treated with the agent with the protein expression profile of cells not treated with the agent and determining proteins that have altered expression due to the exposure to the agent in the treated cells.
  • the cells may comprise, for example, a variety of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent, and the protein encoded by gene.
  • An additional embodiment includes a method of identifying a number of genes associated with hypersensitivity to an agent which comprises comparing the protein expression profile, where the proteins are encoded by the genes identified as associated with hypersensitivity to the agent, of cells freated with the agent with the protein expression profile of the same type of cells from the same subject not treated with the agent and determining proteins that have altered expression due to exposure to said agent in the treated cells.
  • the cells may comprise, for example, a variety of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent, and the protein encoded by the gene.
  • the gene expression profile of multiple genes associated with cellular response to toxic agents are analyzed to determine the association with hypersensitivity of the genes in the profile.
  • an individual can be screened for hypersensitivity to a drag before the drug is administered. Such screenings avoid incidents of hypersensitivity in individuals to whom a drug might otherwise be administered. Alternately, the drag can be given in lower doses to hypersensitive individuals and/or those individuals considered at risk may be closely monitored for adverse reactions to the agent. Avoiding exposing hypersensitive individuals to any given drag or compound, or to a higher than necessary dose or level of the drag or compound, provides cost savings to manufacturers who may produce the drag or compound with an assurance that hypersensitivity reactions will be avoided.
  • the invention also encompasses using the methods, composition and devices disclosed herein for rapid, accurate and inexpensive tests that can be used, for instance, to determine the causative agent in an individual exhibiting symptoms consistent with or indicative of a toxic response or hypersensitivity to various agents. By ascertaining the gene profile of a number of genes associated with particular cells, tissues, organs or systems, the agent eliciting the toxic response or hypersensitivity may be determined and thereon avoided.
  • gene expression analysis might be used to determine the nature of the toxic insult and thus provide treatment.
  • analysis of expression of tox-response genes might aid in the effective diagnosis and treatment of an unconscious child suspected of having been inappropriately exposed to a drag or chemical agent.
  • Gene expression patterns could be useful in determining if the unconscious state were the result of exposure to a soporific agent or one that inhibited mitochondrial function, the treatments of which would be quite distinct.
  • Exemplary genes associated with hypersensitivity whose expression may be screened in order to determine hypersensitivity are provided in whole or in part in Tables 3, 4, 5, 6, 8, 10 and 11. Also provided herein are methods of identifying genes associated with hypersensitivity.
  • Tables 3, 4, 5, 6, 8, 10 and 11 provide a list of exemplary genes from which genes associated with hypersensitivity to a particular agent may be selected. Genes selected from Table 3 and Table 4 are responsive to toxic stimuli and important to the defense or repair of toxic damage. Individuals with significantly altered expression levels of two or more of the genes in Tables 3, 4, 5, 6, 8, 10 and 11 can also show different toxic responses from normal individuals. For a given agent, the expression profile of two or more genes, for example, selected from Tables 3, 4, 5, 6, 8, 10 and 11 can be obtained from a cell, tissue or organ and, a pattern of gene expression predetermined to be associated with hypersensitivity can be established.
  • Genes such as those selected from Tables 3 and 4 are evaluated for differential gene expression, for example in the major toxic target organs in humans and/or rats and mice.
  • Examples of genes in which differential expression is indicative of toxicity or hypersensitivity in specific organs or systems such as liver (hepatic), kidney (renal), lung (pulmonary), central nervous system (neural), heart (cardio) and immune system are shown in Table 5.
  • Figure 1 shows the pattern of gene expression of approximately 250 genes in the liver when the subject received a relatively high dose of streptozotocin.
  • Samples including for instance, blood, urine, serum or tissue, from individuals known to be hypersensitive to streptozotocin can be obtained after the subject is treated with streptozotocin. Alternately, for example, samples may be from untreated individuals known to be hypersensitive to streptozotocin and the samples may then be treated in vitro with streptozotocin. The samples are then examined to identify genes associated with hypersensitivity.
  • This may show, for example, highly exaggerated expression of toxic response genes and/or patterns of induction or repression of genes in treated individuals or upon in vitro treatment of the sample with streptozotocin compared to individuals who are not hypersensitive or sample which is not treated with streptozotocin.
  • streptozotocin is an example of a bulky alkylating agent
  • individuals who are hypersensitive to streptozotocin may be tested for hypersensitivity to compounds with similar toxic properties, such as bulky alkylating agents, such as merbarone and carmustine.
  • Genes whose levels of expression change in response to toxic stimuli may be evaluated. Examples of genes with expression changes in response to toxic stimuli are listed in Tables 3 and 4.
  • the genes in Table 3 and Table 4 have been shown to be induced in either cell lines, primary cells, tissues or tissue slices, from human or animal origin.
  • the GADD 153 gene has been shown to be induced in many human cell lines upon exposure to radiation.
  • the environmentally important compound trichloroethylene was recently demonstrated to cause induction of several genes, including c-Myc and c-Jun in mice exposed to low toxic levels for 24 hr. Tao et al.(J Biochem Mol Toxicol) 13(5):
  • Many compounds are toxic at a high enough concentration. For example, while most individuals might experience extreme tachycardia after receiving a very high dose - 20 times normal - of a drag, they experience no such effects at recommended doses. The hypersensitive individual would experience extreme tachycardia at the recommended dose or at a lower than normal dose. A hypersensitive individual might also experience a qualitatively distinct toxic response to a compound, not just the same response that a normal individual would experience at high doses. For example, the hypersensitive patient might experience extreme dizziness, a side effect not reported by individuals even at high doses.
  • Agents to which individuals may be hypersensitive, and for which hypersensitivity can be determined may include, for example, drags, industrial chemicals, household or other chemicals, including those in the workplace. Examples of drugs and industrial chemicals for which a sub-population is hypersensitive are listed in Tables 1 and 2. As a further example, individuals who are employed in manufacturing or other environments which expose them to a variety of agents may be screened for agents to which they might come into contact. Individuals, or for example, a subset of workers, who are hypersensitive to the agents can then be identified. Hypersensitivity to other agents also may also be determined, such agents including, but not limited to biological agents such as naturally occurring organic compounds, including proteins, saccharides and lipids.
  • Exemplary pharmaceutical agents include, for example, tienilic acid, halothane, dihydrazine, diclofenac, fialuridine, carbamazepine, TrovanTM (trovafloxacin), SeldaneTM (terfenadine), hismanol, dihydrolazine, warfarin, phenytoin, omeprazole, diazepam, haloperidol, perphenazine, perhexiline, phenformin, tolbumamide, penicillin, clozapine, aminopurine, quinidine and remoxipide.
  • Table 1 lists additional agents for which there are individuals who demonstrate hypersensitivity.
  • examples of other chemicals include industrial chemicals, such as paint, volatile organic compounds (VOCs), solvents, adhesives, pesticides, herbicides, perfumes, aerosols, cleaning compounds and synthetic polymers such as textiles.
  • Hypersensitivity to an agent such as a drag
  • Hypersensitivity may also be determined based on the ability to identify the underlying molecular basis for the toxicity of specific drags. Hypersensitivity can also be determined by examining the gene expression of hypersensitive and normal individuals.
  • methods are provided wherein literature reports on the expression levels of single genes in response to a single agent are collected, for example, in a database, and then analyzed to establish patterns of expression that can be conelated to hypersensitivity.
  • large amounts of data can be collected and analyzed, for example by software means.
  • Matrix Express and Chem Profiler (Phase- 1 Molecular Toxicology, Santa Fe, NM) accommodate capture and analysis of gene expression profiles. For example, it allows identification of induced genes from the total set of genes measured using a number of criteria; for example, statistical significance, twofold, and 1.5 X the standard deviation.
  • the software also allows the search of other profiles and determines the commonality between subsets, ranking profiles by several measures of similarity, for example, using all or a subset of the genes.
  • Experiments include both in vivo and in vitro responses to agents, for example, the exposure of eukaryotic, mammalian or human cells, and animals to agents listed in Table 7.
  • agents for example, the exposure of eukaryotic, mammalian or human cells, and animals to agents listed in Table 7.
  • One ultimate benefit of this exercise is to reduce the need for animal testing. Each agent is tested at several concentrations and time points.
  • the toxicology of an agent is evaluated by measuring toxic insult by detecting observable changes in organ or system appearance and/or function, at the micro- or macroscopic levels. For example, a drag may cause changes in fatty acid metabolism in liver hepatocytes. This in turn causes observable changes in liver appearance, such as a specific toxicological outcome refened to as fatty liver.
  • a drag may cause changes in fatty acid metabolism in liver hepatocytes. This in turn causes observable changes in liver appearance, such as a specific toxicological outcome refened to as fatty liver.
  • a drag may cause changes in fatty acid metabolism in liver hepatocytes. This in turn causes observable changes in liver appearance, such as a specific toxicological outcome refened to as fatty liver.
  • a specific toxicological outcome refened to as fatty liver.
  • genes that are differentially expressed in response to toxic injury are evaluated for use as genes associated with hypersensitivity in accordance with the present invention.
  • genes that are differentially expressed in total across cell, organ and tissue types in humans, in particular in response to toxic insult may be evaluated to determine which genes have expression that is linked to hypersensitivity in an individual.
  • Organs are composed of tissues, which in rum are composed of various cell types. There is a core set of genes whose products are involved in functions essential to all cells, and whose expression is shared by most human cell types. In addition to these common core genes, each cell type expresses a set of genes that is unique to that cell type. When animals, including humans, are exposed to chemicals that cause damage to one or more organs, cells that comprise those organs attempt to mitigate or repair that damage by turning on genes that encode toxic-damage defense or repair proteins. The specific set of genes that cells induce is dependent upon the type of damage or toxic threat caused by the compound and upon which organs are most threatened. In addition to the genes that are induced to deal with the specific toxic threat, there may be genes which encode functions that are not needed nor appropriate under conditions of toxic injury.
  • both the up- and down-regulation of genes can be measured in order to understand the molecular response to that compound, and the linkage of gene expression to hypersensitivity.
  • the pattern of differential gene expression within the toxic target organs can be limited to a relatively small number of genes, and may be very specific to both the organ being threatened and the type of damage.
  • Such genes may be analyzed to determine which genes are responsible for hypersensitivity, for example, within a certain organ.
  • Such genes may be analyzed to identify subsets of genes that are associated with hypersensitivity to certain agents.
  • the measurement of gene expression patterns is useful because many factors can affect the level of transcripts of toxicity genes, including mutations in the regulatory regions of genes, mutation in transcription factor that control the gene(s) of interest, and gene duplications and deletions.
  • genes associated with changes in expression levels due to adverse stimuli or toxic insult include, for example, genes which respond to the presence ofa compound, and genes which respond to damage caused by a compound at, for example, the protein, nucleotide, macromolecular, membrane, cell, tissue, organ or system level. For example, certain proteins either prevent or repair toxic cellular injury. Individuals who do not express the appropriate gene profile will suffer greater damage from toxic compounds through a lack of repair enzymes.
  • Toxic responses can be measured by pathological changes, for example, at the protein, nucleotide, cell, tissue, organ or system level. These pathological changes can be associated with differential gene expression of at least two genes. In addition, and the conespondence between the pathological change and the differential gene expression can be established. At the concentration where pathological outcomes are observable, gene expression changes are specific and causally related to the outcome. For example, compounds that cause peroxisome proliferation as observed in the electron microscope, such as WY 14,643 (Sigma Chemicals; St. Louis, MO), a common toxicological compound known in the art, turn on genes causally related to peroxisome proliferation (See Figure 4).
  • Genes which may be identified and tested for their association with hypersensitivity to a certain agent include a variety of genes known in the art that are induced in mammalian or eukaryotic cells or cell lines exposed to high concentrations of chemicals. Genes associated with toxicological response that can be identified for predicting different types of hypersensitivity to different agents include, for example, those genes described in: Cattell (Semin. Nephro.) 19(3):277-87 (1999); Schnabel, M. et al. (Int. J. Mol. Med.) l(3):593-5 (1998); Cruse et al. (Carcinogenesis) 20(5) 817-824 (1999); Fogg, S. et al.
  • a 'temporary' knock-out of the cyclophilin-A gene in mice was made by injecting an anti-sense RNA against the cyclophilin A gene in rat neonatal cardiomyocytes.
  • the expression level of the cyclophilin A gene was reduced by 93% and animals freated were hypersensitive to the toxic effects of t-butylhydroperoxide. Doyle et al. (Biochem. J.) 341( l):127-32 (1999). Humans who show depressed levels of cyclophilin A gene expression are expected to be hypersensitive to the toxic effects of t- butylhydroperoxide and other compounds that form active oxygen radicals.
  • PON1 serum paraoxonase
  • OP organophosphate
  • One polymorphism (Argl92 isoform) hydrolyzes diazoxon, soman and sarin slowly. Costa et al. (Chem. Biol. Interact) 119-120: 429-38 (1999).
  • Genes associated with hypersensitivity can be selected from those in Table 3, which are induced by toxic damage and have important physiological roles in responding to toxic stimuli.
  • Rettie et al. (Epilepsy Res.) 35(3):253 (1999) showed that humans carrying a polymorphism that decreases expression of the CYP2C9 gene are very sensitive to compounds such as phenytoin and (S)-warfarin.
  • the data demonstrate that the
  • CYP2C9*3 polymorphism gene product retains only 4-6% of the metabolic efficiency of the wild-type protein CYP2C9*1 towards phenytoin and (S)- warfarin. Individuals who show dramatically reduced expression of the normal CYP2C9*1 could show the same hypersensitivity to these drags.
  • Several factors can affect the basal and induced levels of expression of these genes. For example, mutations or polymorphisms that affect the promoter region of tox- response genes can cause hypersensitivity to compounds. For example, several polymorphisms have been identified in the promoter region of the human HLA-DQAl gene that affect the levels of mRNA and thus protein levels of the HLA haplotype. Indovina, P. et al. (Hum.
  • TPMT expression displays genetic polymorphism with 10% of individuals having intermediate and one in 300 undetectable levels.
  • polymorphisms comprise a significant percentage of the population.
  • a genetic polymorphism in the metabolism of the anticonvulsant drag S-mephenytoin has been attributed to defective CYP2C19 alleles.
  • This genetic polymorphism displays large intenacial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. Ibeanu et al. (J. Pharmacol. Exp. Ther.) 286(3): 1490-5 (1998).
  • UGTl A UDP- glucuronosyltransferase locus
  • a knock-out mutant has been created in mice that destroyed the function ofa single gene, the au-beta 6 gene. The resulting animals showed altered basal expression of 101 genes in lung epithelial cells.
  • Single mutations in any one of hundreds of key toxicity genes can potentially cause differential basal levels of expression of many additional genes. It may be the altered expression of these genes that render the cell, or organism sensitive to toxic stress, not the initial mutation by itself.
  • Genes associated with hypersensitivity to an agent may be identified in a variety of ways experimentally. Generally the expression of genes that are differentially expressed in total across cell, organ and tissue types in humans, in particular in response to toxic insult is evaluated to determine genes associated with hypersensitivity in an individual.
  • a method of identifying genes associated with hypersensitivity to an agent comprises comparing the gene expression profile of cells treated with an agent with the gene expression profile of untreated cells, and determining genes in the treated cells that have altered expression due to the treatment, thereby to identify one or more genes associated with hypersensitivity to the agent.
  • the cells may comprise one or more different cell types, wherein each said cell type comprises a gene associated with hypersensitivity to the agent. Alternately, the cell types are derived from a single tissue or organ.
  • Exemplary cell types are those derived from a specific organ , cell or tissue, such as kidney, liver, lung, heart, breast, lymphocytes, neuronal cells, skin, or intestine, such as HepG2, Caco-2, MCF-7, Jurkat, Daudi, HL-60, MCL-5, SKBR-3, SKOV-3, PC-3, WISH and HeLa.
  • a specific organ , cell or tissue such as kidney, liver, lung, heart, breast, lymphocytes, neuronal cells, skin, or intestine, such as HepG2, Caco-2, MCF-7, Jurkat, Daudi, HL-60, MCL-5, SKBR-3, SKOV-3, PC-3, WISH and HeLa.
  • Another method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent comprises comparing the gene expression profile of multiple cell types of an individual known to be hypersensitive to an agent with the gene expression profile of said cell types in an individual known not to be hypersensitive to the agent; and identifying genes from said multiple cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent.
  • An alternative to this method comprises, comparing the gene expression profile of multiple cell types of an individual known to be hypersensitive to an agent before treatment with the agent with the gene expression profile of multiple cell types of the hypersensitive individual after treatment with the agent, and identifying genes from the multiple cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent.
  • Different types of toxic insult lead to different patterns of gene expression changes in normal, as well as in hypersensitive individuals. Since substantially all compounds elicit toxicity at a high enough dose, the mechanisms of drag toxicity in normal individuals has been well examined. Genes that cells induce to combat the toxic effect of various compounds are important for anti-toxicity for each compound. Patterns of gene expression of these genes in individuals who show hypersensitivity to a given compound that differ from the pattern of differential expression of normal individuals, with or without treatment can be identified. Using these methods, sets of genes that have characteristic expression in hypersensitive individuals that differs from normal individuals may be identified.
  • Subsets of genes and expression profiles thereof that can be used to identify hypersensitive individuals are identified as follows.
  • a technique such as amplified fragment length polymorphism (AFLP) or serial analysis of gene expression (SAGE), which are known in the art, is used to compare gene expression profiles from treated and untreated human cells.
  • the agent is administered at a toxic dose.
  • This procedure identifies all candidate genes within the cells that respond to the toxic stimuli posed by a particular agent.
  • the method further comprises using a technique, such as AFLP or SAGE, which are known in the art, to compare the gene expression profiles from treated and untreated normal cells. This step would identifies all genes within an individual that respond to that agent. It also permits investigators to understand the normal expression range of individuals who are not hypersensitive.
  • a technique such as AFLP or SAGE, is used to compare gene expression profiles for samples from treated and untreated hypersensitive individuals or cell cultures derived therefrom. This step identifies all genes within hypersensitive individuals that respond to the treatment by that agent. It also allows investigators to understand the expression range of hypersensitive individuals. This permits identification of the genes that were differentially expressed in all of the above experiments, thus eliminating genes associated with therapeutically beneficial effects and individual variation in expression of genes unrelated to the compound. The expression of these genes can then be measured in a larger population of normal and hypersensitive individuals using, for example gene anays, RT-PCR or other techniques known in the art to confirm the conelation between those genes identified in the above procedures and hypersensitivity observed in particular individuals.
  • Gene expression responses to toxic stimuli can be analyzed using a database of information.
  • the first method is to determine which genes are induced and what is their function. For example, if all genes induced by a compound are regulated by DNA damage, the interpretation is that the compound causes DNA damage. This interpretation requires a database about the function and regulation of all genes in the database.
  • Another method of interpretation is to determine whether the gene expression pattern induced by a second compound is similar to that induced by a compound, the toxicity of which is well- characterized. This approach to interpretation requires an extensive database of gene expression profiles generated from well-characterized compounds. Table 7 shows a partial list of well-characterized compounds for which gene expression data has been generated.
  • the methods of gene expression analysis discussed herein can be performed using a computer system with computer code suitable for accessing and comparing the gene expression profile determined according to the methods of this invention. Suitable software will also rank the results of these analyses. Computer code suitable for these purposes can be programmed by a person skilled in the art. Exemplary software and a gene expression profile database related to toxicology are commercially available from Phase- 1 Molecular Toxicology, (Santa Fe, NM), for example, Chem ProfilerTM and Matrix ExpressTM.
  • Drags known to elicit Steven Johnson Syndrome and TEN and less severe forms of skin allergy include navirapine, dapsone, acebutolol, trimethoprim, sulfasalozine, sulfacetamide, sulfadiazine, sulfamethoxizole, sulfasoxazole, sulfamethizole cotrimoxazole, amoxacillin, phenytoin, sulfonamide and penicillin.
  • genes whose expression in CD8 T Cells and keratinocytes is likely to identify hypersensitive individuals include: inducible NOS, Ki-67, Transglutaminase-1, IL-1, FASL, TNF -alpha, CD 1 lb/CDl 8, p75-R-TNF (TNF Receptor), IL-6 receptor, G-CSF receptor, HSP-70, INF- gamma, ICAM-1, VCAM-1, ECAM-1, and TGF-beta.
  • Example 1 Identification of Genes Associated with Hypersensitivity and Screening of Subjects Prior to Drag Administration
  • HaldolTM haloperidol
  • haloperidol is determined in vomerophils from both normal and hypersensitive subjects when exposed to high concentrations of HaldolTM (haloperidol).
  • the gene expression profile from untreated and treated cells is compared using for example, AFLP, a microanay of the genes listed in Tables 3 and 4, or SAGE, to identify genes that vary as a function of toxicity and vary as a function of hypersensitivity to the HaldolTM (haloperidol).
  • AFLP a microanay of the genes listed in Tables 3 and 4, or SAGE
  • gene expression from clinical samples from a patient population exposed to HaldolTM (haloperidol) or a placebo is measured.
  • the clinical samples are provided by the manufacturer of HaldolTM (Hoechst Marion Roussel).
  • Genes are identified that co-varied with the hypersensitivity status. Additional clinical samples are blinded and provided by the manufacturer which includes samples from normal and hypersensitive subjects.
  • prediction of the hypersensitivity status is based upon gene expression profiles.
  • the level of accuracy of the prediction or conect identification of hypersensitivity is determined by monitoring patients over time to see if those predicted to develop agranulocytosis indeed did so. This empirical approach is then be extended to other drags and other drag manufacturers.
  • Example 2 cDNA Probe Production A fluorescent dye labeled cDNA probe complementary to the mRNA component of cellular RNA harvested from cells exposed to toxicologic challenge is produced by this protocol, which is designed to produce sufficient Cy3 labeled probe from one experimental sample, and Cy5 labeled probe from one confrol sample, to develop one microanay slide.
  • the procedure is scalable to easily accommodate, for example, 16 samples. This will produce sufficient probe mixtures for at least 8 microanay slides.
  • General procedures as described, for example, in Gerard et al. (Focus®) 14:91 (1992); Kotewitcz et al. (Gene) 35: 249 (1985); and Gerard et al. (DNA) 5: 271 (1986) are utilized.
  • cDNA probes may be used in an assay for detecting expression of genes associated with hypersensitivity to an agent.
  • microanay slides are provided that contain ssDNA sequences, or targets, from a number of toxicologically relevant genes.
  • the microanay slides may be 3"x 1" glass microscope slides comprising an anay of micron-scale spots of ssDNA sequences on the upper face.
  • the DNA may be bound to the slide using covalent linkage chemistries known in the art.
  • Total RNA from cells contains mRNA species that are homologous to these sequences.
  • Total RNA high quality refers to substantially total cellular RNA.
  • RNA is very labile, special care must be taken to insure that it is of sufficient integrity at the time of use as template in the production of probe.
  • the level of these mRNA species is proportional to the degree of induction of the gene by the agent under study.
  • This protocol describes the production of fluorescent labeled cDNA probe from the total RNA of cells which have either been exposed to the agent under study, or are serving as a non-treated control. These probes are then pooled and hybridized to the microanay slide.
  • the experimental and control probes are distinguishable because the Cy3 and Cy5 labels fluoresce at different wavelengths.
  • the degree to which each probe binds to a specific gene sequence on the slide reveals the level of induction of that gene in the cells exposed to the agent under study.
  • Steps are performed at room temperature unless otherwise specified. Work areas are cleaned and swabbed with RNase Zap. Gloves are worn at all times.
  • RNase RNA specific endo-and exo-nucleases
  • Standard cleaning and/or autoclaving will not remove or inactivate it. Therefore all materials contacting the samples must be known RNase-free.
  • All water, including for buffers, must be DEPC-treated.
  • DEPC treatment consists of an autoclaved solution of 0.1% Diethyl pyrocarbanate in de-ionized water.
  • RNA template in water is implemented by adjusting mRNA to a concenfration of 2 ⁇ g/7 ⁇ l or total RNA to a concenfration of 10 ⁇ g/7 ⁇ l for each sample in a standard microfuge tube. If concentration adjustment requires dehydration in the SpeedvacTM, 1 ⁇ l Anti-RNase is added prior to dehydration.
  • the reaction solution is prepared by adding 4 ⁇ l of stock anchored oligo dT per tube, heating at 70°C for 10 minutes in a heat block, spinning 5 seconds in microfuge, and placing on ice for 2 minutes. The following is then added to each tube:
  • the tube then is incubated at room temperature for 10 min.
  • the dCTP is added to limit the concentration of Cy dCTPs inco ⁇ orated. Due to the size of the Cy dCTP, the polymerase will fall off the template if more than two are inco ⁇ orated in a row.
  • 1 ⁇ l SuperScriptll is added to each tube, and the contents mixed gently.
  • the tube then is incubated for 1.5-2 hr. at 45°C in a heat block, keeping the reaction protected from light.
  • the fluorescent dyes Cy3 and Cy5 are sensitive to light. Excessive exposure during processing will reduce the intensity of emission upon final scanning.
  • ethanol precipitation is implemented by adding to each tube 46 ⁇ l of water, 34 ⁇ l of 7.5M ammonium acetate and 220 ⁇ l of 95% EtOH, and then incubating at -80°C for 15-20 min. If desired, procedure may be interrupted at this point. The sample may be stored at -80°C for up to 7 days. The tubes are loaded in centrifuge with orientation of lid noted, centrifiiged for 15 min at 20800 x g, and the supernatant discarded, to obtain a visible pellet (pink for Cy3, blue for Cy5).
  • the pellet is washed by adding 750 ⁇ l 70% EtOH per tube and vortexing briefly, centrifuging at 20800 x g for 10 min, decanting and discarding the supernatant, centrifuging the pellet and optionally gently removing remaining EtOH with a pipette, while being careful not to loosen the pellets.
  • the pellet is allowed to dry for 10 min. at room temp, but not over drying by using a vacuum, and resuspended in 40 ⁇ l water.
  • cDNA/mRNA hybrid is denatured by incubating at 95 °C for 5 min. in a heat block. The tube then is spun 5 seconds in microfuge.
  • the labeled cDNA probe is purified in an adaptation of the procedure described on page 18 of the QIAquick Spin Handbook, (1997) Qiagen®.
  • 200 ⁇ l of Buffer PB is added to each 40 ⁇ l probe solution, the QIAquick spin columns are placed in 2 ml collection tubes, and the samples are applied to the QIAquick columns and centrifiiged at 10,000 x g for 2 min. The flow-through is discarded and QIAquick columns replaced into the same tubes.
  • 750 ⁇ l Buffer PE/ETOH is added to each column, and the column incubated for 1 min. at room temp.
  • the column is centrifiiged at 10,000 x g for 2 min., and the supernatant discarded. The wash is repeated. QIAquick columns are placed back in the same tubes, and centrifiiged for an additional 1 min at maximum speed with tube lids open. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
  • QIAquick columns are placed in clean 1.5ml microfuge tubes.
  • To elute the cDNA probe 40 ⁇ l (+/- 1 O ⁇ l) Alk. Water is added to the center of each column.
  • the tubes are incubated for 1 min, centrifuge at 6000 x g for 1 min., and the elution steps repeated once into same tube.
  • the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound cDNA.
  • each sample is put in ⁇ 80 ⁇ l of EB buffer, and transfened to one well of a 384 well plate.
  • Scanning including the measurement and recording of the type and degree of fluorescence from each spot on a processed microanay slide, is accomplished in a confocal laser scanning fluorimeter.
  • the fluorimeter is set to the appropriate excitation emission frequencies and records the level of emission for the sample.
  • the exposure time and intensity is controlled, because exposure of the label to strong light incrementally reduces its fluorescent activity. Values from this procedure are the result of many variable factors. Therefore it is preferable to compare to an archive of values produced from the same procedure and equipment.
  • the Cy3 labeled experimental probe is combined with the Cy5 labeled confrol probe. If a control requires multiple reactions, they are combined prior to aliquoting equal amounts to the experimental samples.
  • the combined probes are concentrated to ⁇ l ⁇ l in a Speedvac at a temperature not exceeding 45°C. If the probe is not used immediately, 10 ⁇ l water is added and it is stored at 4°C.
  • Example 3 Determination Of Gene Expression Changes Associated With Toxicity To determine genes useful for identifying patterns of genes associated with toxicity, animals were exposed to concentrations of selected compounds that elicit peroxisome proliferation, a type of liver toxicity. Treatments were with WY 14,643, gemfibrozil and clofibrate in Sprague Dawley rats. Each compound was administered in 1% carboxymethycellulose/0.2% Tween 80 by oral gavage daily for 14 days. Administered doses were to three animals per dose per time point as follows; WY 14,643, 40 mg/kg/day; gemfibrozil, 24 mg/kg/day and 100 mg/kg/day, and clofibrate 40 mg/kg/day and 250 mg/kg/day.
  • hepatocyte growth factor receptor gene New genes associated with and predictive of toxicity were identified. Different types of damage to the liver cause the formation of dead and dying hepatocytes, which the liver replaces to maintain its function. Induction of the hepatocyte growth factor receptor gene by toxic stimuli in both rats and humans was examined. When several nitrosoureas including streptozotocin, carmustine and MNU were used to determine gene expression profiles, all of these compounds induced several genes in common. These compounds are all known to form covalent adducts to the DNA in liver and liver cells. All compounds, for example induce both the hepatocyte growth factor receptor gene and the glutathione fransferase gene.
  • Figure 1 shows the gene expression profile in the liver of male Sprague-Dawley rats when freated with the hepatotoxicant streptozotocin.
  • the probe for the hepatocyte growth factor receptor gene was created by cloning at least a 250 base-pair section from the 3' coding region of the gene starting with total genomic DNA. The fragment was derived by PCR from genomic DNA using two primer with appropriate linkers for insertion into a plasmid vector. A single stranded probe complementary to the cDNA sequence was attached to a glass slide anay using a polyamine attachment.
  • an example of creation of a specific probe for the hepatocyte growth factor receptor is as follows.
  • the first step in the process is obtaining the sequence for the gene.
  • the search for gene sequence is performed using the NIH National Center for Biotechnology Information website using Genbank (http://www2.ncbi.nlm.nih.gov/genbank/query_form.html).
  • Genbank http://www2.ncbi.nlm.nih.gov/genbank/query_form.html.
  • the accession number for the rat hepatocyte growth factor receptor gene is X96786.
  • the sequence information is copied to a Microsoft Word file. Intron sequences are then removed, if present, as well as numbers and white spaces.
  • PCR primer design software program such as Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi).
  • Primers are selected that optimally have a T m in the range of 60°-63°C.
  • the optimal length of the gene fragment is 500 bp. Shorter fragments are chosen if the starting sequence is shorter than 500 bp.
  • the BLAST search software searches for other DNA sequences that are homologous to the target sequence and ranks these sequences according to the amount of homology. This ensures that the chosen gene fragment sequence will not cross-hybridize with a gene sequence other than the desired sequence.
  • PCR primers are ordered and an attempt is made to isolate the gene fragment from a cDNA library that is created by reverse transcription of RNA from either a cell line(H4IIE) or rat tissue. Upon identification of a PCR band of the conect size, the PCR product is cloned into a vector (TA cloning vector, Invitrogen Co ⁇ ., Carlsbad, CA).
  • a bacterial mini-prep is performed to amplify and isolate the plasmid containing the gene fragment of interest.
  • the region of the plasmid containing the gene fragment is then sequenced. If this sequence matches the original target sequence, the target sequence of this clone is amplified by PCR, purified (Wizard system, Promega Co ⁇ ., Madison, WI), quantified, and used for spotting.
  • the probe refers to a population of cDNAs bearing fluorescently active ligands which are produced from the mRNA of the cells under examination, while "probe mixture' refers to a mixture of two or more populations of cDNA.
  • the cDNAs may also be labeled with a variety of ligands, such as fluorescently active ligands, radioisotope ligands or biotinylated ligands.
  • Figure 2 is a graph showing the results, which indicated a very strong conelation between the induction of the glutathione fransferase and hepatocyte growth factor receptor genes. Co-induction thus shows conelation to focal cell death occuning in the liver.
  • Palmitoyl fransferase Epoxide hydrolase, Famesol receptor, Lipoprotein lipase precursor, and MDM-2 have never been reported or previously known to be induced by cardiotoxicity.
  • doxombicin a profile of gene expression characteristic of the cardiotoxin, doxombicin was obtained.
  • Genes thus identified as having altered expression in the presence of cardiotoxin are significant, because individuals with diminished or altered expression of the induced genes may potentially be hypersensitive to the toxicity of doxombicin. Such hypersensitivity could manifest itself at the molecular level as altered induction of these genes as well as a shift in the dose-response curve such that the same genes would be induced at lower concentrations.
  • differential display Three different methods, differential display, microanay technology, and Taqman® assay were used to determine genes associated with hypersensitive reaction to penicillin. Seven self-described penicillin-sensitive individuals and six individuals self-described to have normal reaction to penicillin were tested by differential display. Six self-described penicillin-sensitive individuals and six individuals self-described to have normal reaction to penicillin were tested by microanay technology.
  • Lymphocyte culture Six individuals self-described as penicillin sensitive and seven individuals self- described as having normal reaction to penicillin were used to determine potential hypersensitive reactions to penicillin in humans.
  • Peripheral blood leukocytes PBL
  • PHA Peripheral blood leukocytes
  • One group was exposed to penicillin in vitro for 24 hours and the other group was not exposed to penicillin as a control group.
  • penicillin G is known to elicit an immune response in peripheral blood of individuals with proven penicillin G allergy.
  • RNA from select individual from both groups (treated and untreated) of cultured lymphocytes was isolated as follows. Total RNA of high quality and high purity is isolated from cultured cells by using Qiagen QIAamp® RNA blood mini kit and 2- mercaptoethanol. RNA degradation by RNases is not desirable when synthesizing fluorescent cDNA for hybridization with the penicillin anay. Precautions are taken to minimize the risk of RNA degradation by RNases by wearing gloves, treating work areas and equipment with a RNase inhibitor, for example, RNase Zap (Ambion® Products, Austin, TX) and keeping samples on ice. This total RNA isolation technique is based on a Qiagen QIAamp®RNA blood mini kit and is used with some modification for human lymphocyte cells in a T-75 flask.
  • Cells are checked under the microscope to make sure that they are viable. Cells are dosed with penicillin on the third day in culture (48 hours after introduction of the cells into culture).
  • the resuspended pellet is pipeted into a QIAshredder® column and centrifiiged for 2 minutes at 14,000 ⁇ m in a Eppendorf® 5417C centrifuge.
  • the QIAshredder® column is discarded and 600 ⁇ l of 70% ethanol added to the lysate.
  • the lysate is then pipeted into a QIAamp® spin column sitting in a 2 ml collection tube and centrifiiged for 15 seconds at 14,000 ⁇ m. Any remaining lysate is placed on the same column and the centrifugation is repeated.
  • the QIAamp® spin column with the RNA bound to the column is transfened to a new 2 ml collection tube.
  • the QIAamp® column is transfened to 1.5 ml microcentrifuge tube and 50 ⁇ l of RNase-free water is added to the column and centrifiiged for 1 minute at 14,000 ⁇ m. An additional 50 ⁇ l of RNase-free water is added to the column and centrifiiged for another 1 minute at 14,000 ⁇ m.
  • x 50 x 40 RNA concentration in ⁇ g/ ⁇ l 1000
  • the sample is stored in -80°C freezer.
  • RNA sample cleanings included the following materials: 140 ⁇ l lOx Reaction Buffer, 20 ⁇ l GH-DNase I (RNase free, 10 units/ ⁇ l), 140 ⁇ l 3M NaOAc, and 1 mL DEPC-freated H 2 0.
  • RNA sample cleanings included the following materials: 140 ⁇ l lOx Reaction Buffer, 20 ⁇ l GH-DNase I (RNase free, 10 units/ ⁇ l), 140 ⁇ l 3M NaOAc, and 1 mL DEPC-freated H 2 0.
  • DNase I digestion the following materials were added in order: 50 ⁇ l total RNA (10-50 ⁇ g), 5.7 ⁇ l lOx Reaction Buffer, 1 ⁇ l DNase I
  • RNA pellet was washed with 0.5 mL of 70% ethanol (in DEPC-freated water), and spun for 5 minutes to remove the ethanol. The tube containing the materials were spun again and the residual liquid was removed. The RNA was re-dissolved in 10-20 ⁇ l DEPC-treated water.
  • RNA was quantitated by reading on a spectrophotometer at OD 260 .
  • RNA that is diluted for any pu ⁇ ose, such as quantisation, should not be re-used after freezing and thawing.
  • the integrity of the RNA can be checked by running a few micrograms on a 7% formaldehyde agarose gel and looking for the clear appearance of 28S and 18S rRNA bands.
  • a tube the following ingredients are added: 9.4 ⁇ l dH 2 0, 4.0 ⁇ l 5x RT buffer, 1.6 ⁇ l dNTP (250 ⁇ M), 2.0 ⁇ l of 0.1 ⁇ g/ ⁇ l freshly diluted total RNA that is DNase-free, 2.0 ⁇ l H-T ⁇ M (2 ⁇ M) for a total volume of 19 ⁇ l.
  • the ingredients are mixed well and incubated at 65°C for 5 minutes, 37°C for 60 minutes, 75°C for 5 minutes, and held at 4°C. After the tubes had been at 37°C for 10 minutes, and 1 ⁇ l of Superscript II reverse transcriptase (Life Technologies Inc.) is added to each reaction, and quickly mixed by finger tapping the tubes before the incubation continued. At the end of the reverse transcription, the tubes are spun briefly to collect condensation. The tubes are set on ice for PCR or stored at -20°C for later use. 5. PCR to amplify gel band
  • the following ingredients are used: 10 ⁇ l dH 2 0, 2 ⁇ l lOx PCR buffer, 1.6 ⁇ l DNTP (25 ⁇ m), 2 ⁇ l of 2 ⁇ m H-AP primer, 2 ⁇ l of 2 ⁇ m H-T ⁇ M, 2 ⁇ l RT-mix described above (must contain the same H-T ⁇ M used for PCR), 0.2 ⁇ l ⁇ - 33 p DATP (2000 ci/mmole), 0.2 ⁇ l TAQ DNA polymerase from PE
  • Biosystems for a total volume of 20 ⁇ l The tube containing all these ingredients are mixed well by pipeting up and down and placed in a thermocycler at 95°c for 5 minutes and then amplified for 40 cycles under the conditions of 94°c for 30 seconds, 40°c for 2 minutes, 72°c for 30 seconds and finally held at 4°c until the samples are removed from the thermocycler.
  • a 6% denaturing polyacrylamide gel in TBE is prepared and allowed to polymerize for at least 2 hours before using. Then the gel is run for about 30 minutes before any samples are loaded. It is important for all the sample wells in the gel to be flushed and cleared of all urea prior to loading any samples in the wells. About 3.5 ⁇ l of each sample is mixed with 2 ⁇ l of loading dye and incubated at 80°C for 2 minutes immediately before loading onto the 6% gel.
  • the loading dye is xylene and after the gel is loaded with the samples obtained from the rounds of PCR, the gel is run at 60 watts of constant power until the xylene dye is about 6 inches from the bottom of the gel. Once the power is turned off, the gel is blotted onto a large sheet of exposed autoradiograph film. The gel is covered with plastic wrap and under dark conditions, the gel is placed in a large autoradiograph cassette with a new sheet of unexposed film, marked for orientation, and the film is allowed to be exposed to the gel at -80°C. The exposure period can be anywhere from overnight to
  • bands of interest which show differential expression between penicillin sensitive and normal individuals, are identified by alignment with the developed film and subsequently isolated by cutting the band of interest out of the polyacrylamide gel with a clean scalpel blade. The isolated band is placed in 100 ⁇ l of water and boiled at 95% for 5 minutes.
  • the following procedure was used to clone re-amplified PCR products from differential display.
  • Material which may be used include the PCR-TRAP® Cloning System (GenHunter®).
  • PCR-TRAP® Cloning System GeneHunter®
  • For a 20ul Ligation reaction add in order: lOul dH2O; 2ul 10X ligase buffer; 2ul Insert-ready PCR-TRAP® Vector; 5ul PCR product; lul T4 DNA ligase.
  • the reaction is mixed well by finger tipping and is briefly spun. Then the reaction is ligated overnight at 16°C. The reaction can then be used directly for transformation or stored at -20°C.
  • the GH-competent cells are thawed in ice water slush for 15 minutes.
  • the appropriate number of 1.5ml microfuge tubes are labeled and set on ice.
  • the cells are quickly mixed by finger tipping and are divided into lOOul aliquots into each 1.5ml microfuge tube.
  • the remaining competent cells are immediately re-frozen for future use.
  • the ligation tubes are spun briefly to collect condensation. About lOul of each ligation mix is added to an above tube containing the competent cells and mixed well by finger tipping and incubated on ice for 2 minutes. About 0.4ml of LB medium is added and the cells are incubated at 37°C for 1 hour.
  • the LB-Tet plates are warmed at 37°C for 1 hour before plating. After vortexing briefly, about 200ul of cells are plated on an LB-Tet plate (containing lOug/ml of tetracycline). For the lacZ control insert, about 200ul of cells are added to the plate. Then 30ul of X-gal is added to the middle of the cells and the cells are immediately spread onto the LB-Tet plate. Unplated cells can be stored at 4°C if replating is needed within 1 week.
  • the plate surface is dry, the plate is incubated upside-down overnight at 37°C.
  • the Tet colonies are scored and the plate is save upside-down at 4°C.
  • Three individual Tet resistant colonies are picked for each clone with a lOul pipette tip, placed in labeled sterile culture tube containing 3ml of LB broth and grown overnight at 37°C.
  • Plasmid DNA was isolated using the Qiagen Qiaprep Miniprep kit. PCR was used to check for inserts in the plasmids. For each colony the following PCR reaction mixture was set up: dH2O lO ⁇ l lOxPCR buffer 2 ⁇ l dNTPs (250 ⁇ M) 1.6 ⁇ l
  • the PCR parameters were 94°C for 30 sec, 52°C for 40 sec, 72°C for 1 min for 30 cycles followed by 5 min extension at 72°C and a final incubation at 4°C. All 20 ⁇ l of the PCR product was analyzed on a 1.5% agarose gel with ethidium bromide staining. Once the positive colonies were identified, they were sequenced by standard methods well-known to a skilled artisan. The sequences were compared to known sequences to determine if the sequence was already known.
  • nucleic acids comprising said novel sequences and fragments thereof as well as amino acid sequences encoded therefrom and fragments thereof. Also provided are nucleic acids that hybridize to said novel sequences under stringent conditions.
  • Such stringent conditions include conditions of a hybridization reaction that allow nucleic acid duplexes to be distinguished based on their degree of mismatch.
  • Means for adjusting the stringency of a hybridization reaction are well-known to those of skill in the art. See, for example, Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Ausubel, et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1996 and periodic updates; and Hames et al, NUCLEIC ACID HYBRIDIZATION: A PRACTICAL APPROACH, IRL Press, Ltd., 1985.
  • conditions that increase stringency include higher temperature, lower ionic strength and absence of solvents; lower stringency is favored by lower temperature, higher ionic strength, and higher concentrations of solvents (for example, formamide or dimethyl sulfoxide).
  • genes identified using the methods disclosed herein include hypothetical protein (HSPC004), UBA3 (UBA3) mRNA, clone CTA-732E4 on chromosome 22ql2.1, ribosomal protein S7 (RPS7), myosin-binding protein C, cardiac (MYBPC3), CGI-51 protein mRNA, latexin mRNA,
  • NADH oxidoreductase subunit MWFE jun B proto-oncogene (JUNB), KIAA0787 protein, fatty acid synthase, polymerase (RNA) II (DNA directed) polypeptide B (140 kD), UbA52 gene coding for ubiquitin-52 amino acid fusion protein, small nuclear ribonucleoprotein 70kD polypeptide (RNP antigen) (SNRP70), isocitrate dehydrogenase 3 (NAD+) gamma (IDH3G), clone 565E6 on chromosome 1 Iql2-lq22.2, hypothetical protein FLJ20436 (FLJ20436), c-Cbl-interacting protein L7a (RPL7A), ribosomal protein L7a (RPL7A), ribosomal protein S21 (RPS21 ), sorting nexin 6 (SNX6), TNF-inducible protein CGI 2- 1
  • CG12-1 BRCA2 gene region chromosome 13ql2-13, CGI-128 protein mRNA, Tu translation elongation factor, mitochondrial (TUFM), KIAA0787 protein, ribosomal protein L13 (RPL13), ribosomal protein L19 (RPL19), clone 245M18 on chromosome 6p21.32- 22.3, clone TCBA00781, chromosome 19 cosmid R26529, tumor suppressing subfransferable candidate 1 (TSSC1), transfe ⁇ in receptor (TFRC), ubiquitin-conjugating enzyme E2D 3 (UBE2D3), putative DNA-directed RNA polymerase III Cl 1 subunit, myosin-binding protein C (cardiac) (MYBPC3), tapasin (NGS-17), CoREST protein (COREST) (KIAA0071 protein), dynamitin (dynactin complex 50 kD subunit) (DCTN-50), alpla-L-
  • Gene expression profiles comprised of 180 genes on the penicillin anay were compared for similarity between six penicillin-normal individuals and six self-identified penicillin- sensitive individuals. Three of the penicillin-sensitive profiles were repeat samples taken at different times. As shown in Figure 6, Samples 6005, 6015, and 6042 are from one individual, and samples 6041 and 6043 are from another individual. Using all genes for comparison, sensitive individuals tend to resemble one another while non-sensitive individuals have little discemable pattern. The one exception is non-sensitive individual 6002, whose profile has some resemblance to the sensitive individuals. In an exploratory analysis, independent-samples t-tests were performed to suggest which genes were differentially expressed between penicillin-sensitive and penicillin-insensitive individuals.
  • Figure 8 shows that the 20 discriminator genes were analyzed for co-regulation, revealing several co-varying groups, as shown in both the similarity matrix and the relevance network grouping.
  • microanay techniques were utilized to determine genes related to penicillin hypersensitivity. The following are methods that were used to prepare microanay for testing for penicillin hypersensitivity. Of 260 potential gel band, 220 were cloned and sequenced. About 180 genes were put on a penicillin anay, made as described below, and 20 discriminator genes (Table 11) were selected related to penicillin hypersensitivity.
  • 4X Master Mix can be made with the following materials:
  • dNTPs was obtained from Pharmacia Ultrapure dNTP set, cat#27-2035-02 (set contains all 4, 1ml each) and Taq Polymerase was obtained from Perkin Elmer N808-0155 (comes with 10X buffer).
  • Template and gene-specific primer mix was made for 2 rows, or 16 wells by utilizing the following materials: 400ul H 2 O, 2.5ul plasmid, 15ul of lug/ul gene specific primer.
  • Plates can be stored at 4°C for up to 48 hours (maybe more) before cycling.
  • Binding Buffer Immediately mix the Binding Buffer and the PCR sample thoroughly by pipetting up and down 10 times with an automatic pipetting device. Mixing should be completed as quickly as possible (within 5 minutes after adding the Binding Buffer to the SuperFilter 100) to minimize the loss of the Binding Buffer due to gravity flow, void splashing the contents from well to well.
  • the genes to be attached to the glass slides are amplified as provided herein.
  • An important modification to the amplification process is the inclusion of amine primers, which can be obtained from any commercial source, i.e. Synthegen, such that a reactive amine group, a derivative thereof, or another reactive group is included in the amplified product.
  • the amplified product is purified by any number of methods disclosed herein and immobilized or "spotted” onto a solid substrate, such as a glass slide, which can react with the amine group on the amplified product and form a covalent linkage.
  • An MD Generation II Anay Spotter main instrument (Molecular Dynamics, 928 East
  • Spotting Chamber Area of spotter enclosed in glass which houses the pins, plates, trays and most spotter machinery.
  • Target A solution of PCR product which the spotter deposits on the slides.
  • N2 Tank 5 ft. high steel gas tank labeled "Nitrogen, Compressed” N2 : The N2 gas from the N2 tank
  • Air Conditioner Kenmore air conditioner installed in window of spotting chamber
  • Humidifier 1 Essick 2000 Evaporative Cooler against the window
  • Humidifier 2 Bemis Airflow with white flexible duck into the Spotter Unit
  • Humidifier 3 Bemis Airflow against the wall
  • Humidifier 4 Kenmore QuietComfort 7 Vacuum Pump: Gast Laboratory Oilless Piston Vacuum Pump Dampbox: The plastic sealable container containing an NaCl / water slurry
  • Materials used for reagent solutions are: Nanopure water, 0.2 M KC1 (1/10 dilution of Stock 2M KCL in water), and 95% EtOH Reagent.
  • the temperature control is adjusted to 60°.
  • the spotter chambers are adjusted to be greater than 39 % relative humidity and less than 65° C.
  • the spotting pins are pre-washed for 20 cycles.
  • the slides are first each blown with N 2 gas for about 2 seconds per side.
  • the slides are inserted into the Spotter following Anay Spotter Run Values.
  • the slides are aligned using a clean nanow rod orienting it on the center right edge of the slide and gently pushed to the left until the slide is aligned vertically against the metal pins.
  • a visual check is done to make sure no more debris had fallen.
  • the humidity is confirmed to be greater than 39% relative humidity.
  • the MD spotter recognizes 16 plates as a maximum for a run and will pause automatically after 8 plates.
  • the MD spotter also advances sequentially to plates in an invariable order and is not programmable to accommodate unique plate sourcing scheme. Therefore, it is important to manually rotate (or shuffle) plates to accomplish the spotting for the canine anays.
  • This blocking procedure is important because it reduces the non-specific background signals.
  • the amounts provided in this protocol are for 19 slides, however, a skilled artisan may make modifications accordingly. More staining dishes and slide racks will be required if more than 19 slides are to be blocked.
  • a clean glass container is obtained and filled with Nanopure H20. The container is placed on a hot plate and heated to a high temperature.
  • a blocking solution is made by adding 2.5 ml of 20% SDS to 500mL blocking solution bottle. The blocking solution is warmed in microwave for 2.5 minutes and checked to determine if the temperature had reached 50°C. If the temperature of the solution is not at yet 50°C, then the solution is warmed in the microwave at 10 second intervals until it reached the desired temperature.
  • One staining dish is placed on an orbital shaker with 4x SSC solution and turned to an agitation speed of 75 ⁇ m.
  • Slides are placed in metal racks and placed in boiling water for several minutes (i.e. 2 minutes). The slides are taken out of boiling water and allowed to cool briefly. The slides are then transfened to staining container containing 4x SSC solution on orbital shaker for several minutes (i.e. 2 minutes), rinsed with nanopure water in a staining container, and then briefly placed in blocking solution for about 15 minutes. After 15 minutes, the slides are taken out of the blocking solution and rinsed three times by dipping into three separate containers with nanopure water each time. The tops of the slides are dabbed lightly with a tissue and the slides are placed in a centrifuge for about 5 minutes at a speed of 1000 ⁇ m.
  • Fluorescence-labeled first strand cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microanay slides spotted with DNA specific for hypersensitivity relevant genes.
  • the materials needed to practice this example are: total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice. The volume of each sample that would contain 20 ⁇ g of total RNA (or 2 ⁇ g of mRNA) is calculated.
  • the amount of DEPC water needed to bring the total volume of each RNA sample to 14 ⁇ l is also calculated. If RNA is too dilute, the samples are concentrated to a volume of less than 14 ⁇ l in a speedvac without heat. The speedvac must be capable of generating a vacuum of 0 Milli-Ton so that samples can freeze dry under these conditions. Sufficient volume of DEPC water is added to bring the total volume of each RNA sample to 14 ⁇ l. Each PCR tube is labeled with the name of the sample or control reaction. The appropriate volume of DEPC water and 8 ⁇ l of anchored oligo dT mix (stored at -20°C) is added to each tube.
  • RNA sample is added to the labeled PCR tube.
  • the samples are mixed by pipeting.
  • the tubes are kept on ice until all samples are ready for the next step. It is preferable for the tubes to kept on ice until the next step is ready to proceed.
  • the samples are incubated in a PCR machine for 10 minutes at 70°C followed by
  • sample tubes 4°C incubation period until the sample tubes are ready to be retrieved.
  • the sample tubes are left at 4°C for at least 2 minutes.
  • Cy dyes are light sensitive, so any solutions or samples containing Cy-dyes should be kept out of light as much as possible (i.e. cover with foil) after this point in the process. Sufficient amounts of Cy3 and Cy5 reverse transcription mix are prepared for one to two more reactions than would actually be run by scaling up the following protocols:
  • the samples are centrifiiged for 15 minutes at 20800 x g (14000 ⁇ m in Eppendorf model 5417C) and carefully the supernatant is decanted. A visible pellet is seen (pink red for Cy3, blue for Cy5). It is a preferable to centrifuge the tubes at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the probe is seen spread on one side of the tube instead of a tight pellet. If the pellet is white or nonexistent, the reaction has not occuned to maximal efficiency.
  • Ice cold 70% EtOH (about 1 ml per tube) is used to wash the tubes and the tubes are subsequently inverted to clean tube and pellet.
  • the tubes are centrifiiged for 10 minutes at 20800 x g (14000 ⁇ m in Eppendorf model 5417C), then the supernatant is carefully decanted.
  • the tubes are flash spun and any remaining EtOH is removed with a pipet.
  • the tubes are air dried for about 5 to 10 minutes, protected from light. The length of drying time will depend on the natural humidity of the environment. For example, an environment in Santa Fe would require about 2 to 5 minutes of drying time. It is preferable that the pellet are not overdried. When the pellets are dried, they are resuspended in 80 ul nanopure water.
  • the cDNA mRNA hybrid is denatured by heating for 5 minutes at 95 °C in a heat block and flash spun.
  • Millipore MAHV N45 96 well plate v-bottom 96 well plate (Costar), Wizard DNA binding Resin, wide orifice pipette tips for 200 to 300 ⁇ l volumes, isopropanol, nanopure water. It is highly preferable to keep the plates aligned at all times during centrifugation. Misaligned plates lead to sample cross contamination and/or sample loss. It is also important that plate carriers are seated properly in the centrifuge rotor.
  • the lid of a "Millipore MAHV N45" 96 well plate is labeled with the appropriate sample numbers.
  • a blue gasket and waste plate (v-bottom 96 well) is attached.
  • Wizard DNA Binding Resin (Promega cat#Al 151 ) is shaken immediately prior to use for thorough resuspension. About 160 ⁇ l of Wizard DNA Binding Resin is added to each well of the filter plate that is used. If this is done with a multi-channel pipette, wide orifice pipette tips would have been used to prevent clogging. It is highly preferable not to touch or puncture the membrane of the filter plate with a pipette tip.
  • Probes are added to the appropriate wells (80 ⁇ l cDNA samples) containing the Binding Resin.
  • the reaction is mixed by pipeting up and down -10 times. It is preferable to use regular, unfiltered pipette tips for this step.
  • the plates are centrifiiged at 2500 ⁇ m for 5 minutes (Beckman GS-6 or equivalent) and then the filtrate is decanted. About 200 ⁇ l of 80% isopropanol is added, the plates are spun for 5 minutes at 2500 ⁇ m, and the filfrate is discarded. Then the 80% isopropanol wash and spin step is repeated.
  • the filter plate is placed on a clean collection plate (v-bottom 96 well) and 80 ⁇ l of Nanopure water, pH 8.0-8.5 is added. The pH is adjusted with NaOH. The filter plate is secured to the collection plate with tape to ensure that the plate did not slide during the final spin. The plate sat for 5 minutes and is centrifiiged for 7 minutes at 2500 ⁇ m. If there are replicates of samples they should be pooled. To semi-quantitatively assess the inco ⁇ oration of fluorescence into cDNA probes and to concentrate probes prior to hybridization, the following material is used: 384 well, 100 ⁇ l assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent).
  • a consistent amount of cDNA is pipeted into the 384-well plate wells because readings will vary with volume. Controls or identical samples should be pooled at this step, if required.
  • the probes are transfened from the Millipore 96 well plate to every other well of a 384 well assay plate (Falcon Microtest). This is done using a multi- channel pipette. For replicate samples that have been pooled, 60 ⁇ l aliquots are transfened into wells of the assay plate.
  • Cy-3 and Cy-5 fluorescence is analyzed using the Wallac 1420 workstation programmed for reading Cy3-Cy-5 in the 384-well format and the data is saved to disk.
  • the typical range for Cy-3 (20 ⁇ g) is 250-700,000 fluorescence units.
  • Cy-5 (20 ⁇ g) is 100-250,000 fluorescence units.
  • Settings for the Wallac 1420 fluorescence analyzer are as follows:
  • Lamp filter D642 samarium slot B7
  • the dry-down process of the probes is as follows. Concenfration of the cDNA probes is highly preferable so that they can be resuspended in hybridization buffer at the appropriate volume.
  • the volume of the control cDNA (Cy-5) is measured and divide by the number of samples to determine the appropriate amount to add to each test cDNA (Cy- 3).
  • Eppendorf tubes are labeled for each test sample and the appropriate amount of control cDNA is allocated into each tube.
  • the test samples (Cy-3) are added to the appropriate tubes. These tubes are placed in a speed-vac to dry down, with foil covering any windows on the speed vac. At this point, heat (45°C) may be used to expedite the drying process. Time will vary depending on the machinery.
  • the drying process takes about one hour for 150 ⁇ l samples dried in the Savant. Samples may be saved in dried form at -20°C for up to
  • hybridization buffer About 30 ⁇ l of hybridization buffer is prepared per sample. Slightly more than is what is needed should be made since about 100 ⁇ l can be lost during filtration.
  • Hybridization Buffer for 100 ⁇ l: • 50% Formamide 50 ⁇ l formamide
  • the solution is filtered through 0.2 ⁇ m syringe filter, then the volume is measured.
  • About 1 ⁇ l of salmon sperm DNA (lOmg/ml) is added per 100 ⁇ l of buffer.
  • Materials used for hybridization are: 2 Eppendorf tube racks, hybridization chambers (2 anays per chamber), slides, coverslips, and parafilm.
  • About 30 ⁇ l of nanopure water is added to each hybridization chamber. Slides and coverslips are cleaned using N 2 stream.
  • About 30 ⁇ l of hybridization buffer is added to dried probe and vortexed gently for 5 seconds. The probe remained in the dark for 10-15 minutes at room temperature and then is gently vortexed for several seconds and then is flash spun in the microfuge.
  • the probes are boiled for 5 minutes and centrifuged for 3 min at 20800 x g (14000 ⁇ m, Eppendorf model 5417C). Probes are placed in 70 °C heat block. Each probe remained in this heat block until it is ready for hybridization.
  • the slide is gently lowered, face side down, onto the sample so that the coverslip covered that portion of the slide containing the anay. Slides are placed in a hybridization chamber (2 per chamber).
  • the lid of the chamber is wrapped with parafilm and the slides are placed in a 42°C humidity chamber in a 42°C incubator . It is preferable to not let probes or slides sit at room temperature for long periods. The slides are incubated for 18-24 hours.
  • all non-specifically bound cDNA probe should be removed from the anay. Removal of all non-specifically bound cDNA probe is accomplished by washing the anay and using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. It is highly preferable that great caution be used with the standard wash conditions as deviations can greatly affect data.
  • the stainless steel slide earners are placed in the second dish and filled with 2X SSC, 0.1%SDS. Then the slides are removed from glass slide holders and placed in the stainless steel holders submerged in 2X SSC, 0.1%SDS and soaked for 5 minutes. The slides are transfened in the stainless steel slide carrier into the next glass dish containing 0.1X SSC and 0.1%SDS for 5 minutes. Then the slides are transfened in the stainless steel canier to the next glass dish containing only 0.1X SSC for 5 minutes. The slides, still in the slide canier, is transfened into nanopure water (18 megaohms) for 1 minute.
  • the stainless steel slide earners are placed on micro-canier plates with a folded paper towel underneath. The top of the slides are gently dabbed with a tissue. Then the slides are spun in a centrifuge (Beckman GS-6 or equivalent) for 5 minutes at 1000 ⁇ m. It is very important that the slides do not air dry, as this will lead to increased background.
  • RNA total or messenger RNA
  • 3DNATM SubmicroTM Expression Anay Detection Kit Genisphere 3DNA 14 Phillips Parkway Montvale, NJ 07645; Kit numbers: K20F00-41 and K20F00-31); Linear
  • RNA for synthesis of cDNA, prepare 2 separate identical reactions for each sample.
  • a PCR or 1.5ml tube combine: 1.5ug lymphocyte RNA in 7ul DEPC treated water (if sample is too dilute, concentrate it in the SpeedVac at room temperature), and 3ul RT Primer. Separate tubes for freated and untreated RNA. Heat mixture to 80°C for 10 minutes, 4°C for 2 minutes. Place samples on ice and add the following: 4ul 5X RT buffer, lul dNTP mix, 4ul RNase free water, and lul Reverse transcriptase enzyme. Gently mix and centrifuge the contents of the tube. Incubate at 42°C for 1.5 to 2 hours.
  • Taqman® RT Reaction Taqman® technology from Roche Molecular System was used in the following manner. The mRNA was converted to cDNA using 3 ⁇ g total RNA and 1.5 ⁇ l random hexamer primers. After a 10 minute incubation at 70°C the following components were added to the reaction mixture: 6 ⁇ l of 5x first strand buffer, 3 ⁇ l 0.1 DTT, 1.5 ⁇ l lOmM dNTPs, 1.5 ⁇ l Superscript enzyme and 6.5 ⁇ l DEPC-treated water. The reaction is incubated for two hours at 45°C and 1 ⁇ l of this reaction is used for the Taqman® assay.
  • Real time PCR can be performed using the Taqman® assay .
  • the method measures PCR product accumulation with a dual-labeled fluorogenic probe.
  • the probes are labeled with 6-FAM on the 5' end and TAMRA on the 3' end.
  • TAMRA is a quencher dye.
  • This assay exploits the 5 '-3' exonuclease property of Taq polymerase.
  • the reporter dye FAM
  • the reporter dye is cleaved by the 5' exonuclease activity of the Taq polymerase and can emit a fluorescent signal. With increasing cycles of amplification more signal is emitted and detected using an ABI 7700 sequence detector.
  • a set of two primers and a fluorogenic probe are designed and synthesized.
  • probes and primers For quantitation of mRNA the best design for probes and primers requires primers to be positioned over exon-intron junctions. This mles out amplification of contaminating genomic DNA.
  • primer and probe sets For initial studies, primer and probe sets have been designed for 13 genes that were up- or down-regulated by penicillin in differential display experiments. The probes and primer sets were tested for their ability to amplify genomic DNA. If genomic
  • FIG. 9 and 10 show results obtained with a penicillin sensitive person as well as a penicillin refractive person.
  • the genes in these figures are as follows: 1 A is Inhibitor of apoptosis protein- 1, 76B is cyclin D2, 142B is Fc-gamma-receptorllA (FCGR2A), 167B is chromosome 16 clone, RP11-296110 198 A is ribosomal protein S24 (RPS24a), 198B is ribosomal protein S24 (RPS24a).
  • the Y-axis refers to levels of gene expression based on ABI Prism 7700 Realitive Quantification Software, in which cDNA levels are measured based on Ct (Cycle Threshold) values between control and treated samples.
  • Protein expression in lymphocytes was studied using two technologies, SDS Polyacrylamide Electrophoresis (SDS-PAGE) and Surface Enhanced Laser Deso ⁇ tion/Ionization Time-of-Flight Mass Specfrometry (SELDI-TOF) of proteins applied to ProteinChips. Differences in protein profiles, treated and untreated, for sensitive and refractive samples were observed using both techniques. The following methods were used: Cell Preparation
  • SDS-PAGE Proteins were electrophoresed using a Bio-Rad MiniProtean gel apparatus, on ReadyGel Precast 4-20% acrylamide gels, using the standard method of Laemmli. For each concentrated lysate, 20 ul sample was mixed with 5 ul 5X SDS sample buffer. The samples were boiled for 10 minutes in the presence of 2-mercaptoethanol and half of each sample was loaded into conesponding wells on two identical gels. Two stains were used to visualize proteins in the replicate gels, Coomassie Blue and Ruby SYPRO (BioRad). Bands were observed directly for Coomassie stained gels, and by fluorescence scanning (Hitachi Scanner) for Ruby stained gels. All gels were dried in cellophane membranes as permanent records stored in (the laboratory notebook).
  • ProteinChips were obtained from Ciphergen Biosystems. Chips containing spots with hydrophobic (H4) and normal phase (NP) chromatographic surfaces were used. For the H4 surface, 1 ul acetonitrile was pipetted onto each spot to pre- wet the C- 18 surface. Nanopure water was used to to wet the normal phase chip. Three microliters of concentrated lysate was added to each spot on replicate chips, with eight spots/samples per chip. The spots were dried at room temperature, then washed with 10% acetonitrile and nanopure water, for the H4 and NP chips, respectively.
  • H4 hydrophobic
  • NP normal phase
  • Washes were performed by pipetting 5 ul wash solution onto each spot, allowing a 5 minute incubation to resolubilize non-specifically bound biomolecules, and pipetting in and out five times prior to removing the wash buffer.
  • Spots were dried under a 100 Watt bulb (placed 2 feet above benchtop). Each spot was then treated with 0.5 ul sinapinic acid (saturated in 50% acetonitrile, 0.5% trifluoroacetic acid), which acts as an energy absorbing "mafrix" to assist laser ionization of proteins. Proteins were detected directly from the chips using a PBS-II mass spectrometer (Ciphergen Biosystems). Spectra were electronically stored in powe ⁇ oint files.
  • Connexin 32 (gap junction protein) X04325
  • PARP Poly (ADP-ribose) polymerase
  • PCNA Prolifer.cell nuclear antigen
  • Vascular cell adhesion molecule 1 (VCAM-1) M73255
  • Ref-1 redox factor S43127
  • Aldehyde dehydrogenase 1 (ALDH-1) K03000
  • FEN-1 (endonuclease) L37374
  • Organic anion transporter 1 AF057039
  • LIF Leukemia inhibitory factor
  • Urokinase plasminogen activator receptor U08839 c-fms X03663 c-erb B-2 X03363
  • APO-1 cell surface antigen APO-1 cell surface antigen
  • Acid ceramidase actin-binding protein (filamin) (ABP-280)
  • Adenine nucleotide translocator 1 Adenine nucleotide translocator 1
  • CAP Adenylyl cyclase-associated protein
  • Bile salt export pump (sister of p-glycoprotein)
  • BCRP Breast cancer resistance protein
  • CD66e Carcinoembryonic antigen
  • CD44 metalastasis suppressor gene
  • CD64 (Fc gamma)
  • Cytochrome P4504A1 cytoskeletal gamma-actin
  • ECE-1 endothelin converting enzyme
  • ERCC 1 excision repair protein
  • ERCC 5 excision repair protein
  • FEN-1 (endonuclease)
  • Flavin containing monooxygenase 3 for gamma-interferon inducible early response gene (with homology to platelet proteins)
  • GOS24 Zinc finger transcriptional regulator
  • Histone deacetylase 1 HDAC-1
  • hMEF2C myocyte enhancer-binding factor 2
  • HMG-I protein isoform mRNA (HMGI gene), clone 7C
  • Insulin-like growth factor binding protein 1 Insulin-like growth factor binding protein 1
  • Insulin-like growth factor binding protein 2 Insulin-like growth factor binding protein 2
  • Interferon stimulatory gene factor-3 lnterleukin-1 alpha lnterleukin-1 beta lnterleukin-10 lnterleukin-12 lnterleukin-13 lnterleukin-18 lnterleukin-2 lnterleukin-3 lnterleukin-4 lnterleukin-5 lnterleukin-6 lnterleukin-8
  • Keratin 6 isoform K6e Keratin 6 isoform K6e (KRT6E)
  • LIF Leukemia inhibitory factor
  • Lymphoid enhancer-binding factor-1 (LEF-1)
  • Macrophage-stimulating protein (MST1)

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Abstract

L'invention concerne des méthodes permettant d'identifier une hypersensibilité chez un sujet par obtention d'un profil d'expression génétique de gènes multiples associés à l'hypersensibilité d'un sujet susceptible de présenter une hypersensibilité, puis d'identifier, dans le profil d'expression génétique de ce sujet, un modèle d'expression génétique des gènes associés à cette hypersensibilité. Le profil d'expression génétique dudit sujet peut être comparé au profil d'expression génétique d'un individu normal et à celui d'un individu présentant une hypersensibilité. Le profil d'expression génétique du sujet ainsi obtenu peut comprendre un profil de niveaux d'ARN messager ou d'ADN complémentaire. Ce profil d'expression génétique peut être obtenu par utilisation d'un jeu ordonné de sondes d'acide nucléique pour la pluralité de gènes associés à l'hypersensibilité. L'expression de gènes associés à l'hypersensibilité est directement liée à la prévention ou la réparation de lésions causées au niveau tissulaire, organique ou systémique par des substances toxiques. L'invention concerne également des bases de données génétiques, des jeux ordonnés d'échantillons ainsi qu'un appareil destinés à l'identification d'une hypersensibilité chez un sujet.
PCT/US2000/030474 1999-11-05 2000-11-03 Methodes permettant de determiner une hypersensibilite a un agent WO2001032928A2 (fr)

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