EP1100964A1 - Variance de sequences de genes pouvant etre utile pour determiner le traitement d'une maladie - Google Patents

Variance de sequences de genes pouvant etre utile pour determiner le traitement d'une maladie

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Publication number
EP1100964A1
EP1100964A1 EP99935759A EP99935759A EP1100964A1 EP 1100964 A1 EP1100964 A1 EP 1100964A1 EP 99935759 A EP99935759 A EP 99935759A EP 99935759 A EP99935759 A EP 99935759A EP 1100964 A1 EP1100964 A1 EP 1100964A1
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European Patent Office
Prior art keywords
variance
gene
treatment
kinase
variances
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99935759A
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German (de)
English (en)
Inventor
Vincent P. Stanton, Jr.
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Century Technology Inc
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Variagenics Inc
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Publication date
Application filed by Variagenics Inc filed Critical Variagenics Inc
Publication of EP1100964A1 publication Critical patent/EP1100964A1/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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • This application concerns the field of mammalian therapeutics and the selection of therapeutic regimens utilizing host genetic information, including gene sequence variances within the human genome in human populations.
  • the rate of approval of new drugs that enter human clinical trials is less than 20%, despite demonstrated efficacy of said new drugs in preclinical models of human disease.
  • the low response rate in humans is due to genetic heterogeneity in the drug target or the pathway mediating the action of the drug. Identification of the genetic causes of variable drug response would allow more rational clinical development of drugs.
  • many drugs or other treatments approved for use in humans are known to have highly variable safety and efficacy in different individuals. A consequence of such variability is that a given drug or other treatment may be highly effective in one individual, and ineffective or not well tolerated in another individual. Thus, administration of such a drug to an individual in whom the drug would be ineffective would result in wasted cost and time during which the patient's condition may significantly worsen.
  • the present invention is concerned generally with the field of treatment of diseases and conditions in mammals, particularly in humans. It is concerned with the genetic basis of inter-patient variation in response to therapy, including drug therapy. Specifically, this invention describes the identification of gene sequence variances useful in the field of therapeutics for optimizing efficacy and safety of drug therapy for specific diseases or conditions and for establishing diagnostic tests useful for improving the development and use of pharmaceutical products in the clinic. Methods for identifying genetic variances and determining their utility in the selection of optimal therapy for specific patients are also described, along with probes and related materials which are useful, for example, in identifying the presence of a particular gene sequence variance in cells of an individual.
  • the genes involved in the present invention are those listed in a pathway, gene table, list or example herein.
  • the inventors have determined that the identification of gene sequence variances within genes that may be involved in drug action is important for determining whether genetic variances account for variable drug efficacy and safety and for determining whether a given drug or other therapy may be safe and effective in an individual patient.
  • identifications of genes and sequence variances which can be useful in connection with predicting differences in response to treatment and selection of appropriate treatment of a disease or condition.
  • Diseases or conditions are commonly recognized in the art and designate the presence of signs and/or symptoms in an individual or patient that are generally recognized as abnormal. Diseases or conditions may be diagnosed and categorized based on pathological changes. Signs may include any objective evidence of a disease such as changes that are evident by physical examination of a patient or the results of diagnostic tests which may include, among others, laboratory tests to determine the presence of variances or variant forms of certain genes in a patient. Symptoms are subjective evidence of disease or a patients condition - i.e. the patients perception of an abnormal condition that differs from normal function, sensation, or appearance, which may include, without limitations, physical disabilities, morbidity, pain, and other changes from the normal condition experienced by an individual.
  • diseases or conditions include, but are not limited to, those categorized in standard textbooks of medicine including, without limitation, textbooks of nutrition, allopathic, homeopathic, and osteopathic medicine.
  • the disease or condition is selected from the group consisting of the types of diseases listed in standard texts such as Harrison's Principles of Internal Medicine (14th Ed) by Anthony S. Fauci, Eugene Braunwald, Kurt J. Isselbacher, et al. (Editors), McGraw Hill, 1997, or Robbins Pathologic Basis of Disease (6th edition) by Ramzi S. Cotran, Vinay Kumar, Tucker
  • a person suffering from a condition means that a person is either presently subject to the signs and symptoms, or is more likely to develop such signs and symptoms than a normal person in the population.
  • a person suffering from a condition can include a developing fetus, a person subject to a treatment or environmental condition which enhances the likelihood of developing the signs or symptoms of a condition, or a person who is being given or will be given a treatment which increase the likelihood of the person developing a particular condition.
  • tardive dyskinesia is associated with long-term use of anti-psychotics; gastrointestinal symptoms, alopecia and bone marrow suppression are associated with cancer chemotherapeutic regimens, and immunosuppression is associated with agents to limit graft rejection following transplantation.
  • a beneficial change can, for example, include one or more of: restoration of function, reduction of symptoms, limitation or retardation of progression of a disease, disorder, or condition or prevention, limitation or retardation of deterioration of a patient's condition, disease or disorder.
  • Such therapy can involve, for example, nutritional modifications, administration of radiation, administration of a drug, behavioral modifications and combinations of these, among others.
  • drug refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a person to treat or prevent or control a disease or condition.
  • the chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, Hpoproteins, and modifications and combinations thereof.
  • a biological product is preferably a monoclonal or polyclonal antibody or fragment thereof such as a variable chain fragment cells; or an agent or product arising from recombinant technology, such as, without limitation, a recombinant protein, recombinant vaccine, or DNA construct developed for therapeutic, e.g., human therapeutic, use.
  • drug may include, without limitation, compounds that are approved for sale as pharmaceutical products by government regulatory agencies (e.g., U.S.
  • a "low molecular weight compound” has a molecular weight ⁇ 5,000 Da, more preferably ⁇ 2500 Da, still more preferably ⁇ 1000 Da, and most preferably ⁇ 700 Da.
  • the PDR shows that about 45% of patients receiving Cognex (tacrine hydrochloride) for Alzheimer's disease show no change or minimal worsening of their disease, as do about 68% of controls (including about 5% of controls who were much worse). About 58% of Alzheimer's patients receiving Cognex were minimally improved, compared to about 33% of controls, while about 2% of patients receiving Cognex were much improved compared to about 1% of controls. Thus a tiny fraction of patients had a significant benefit. Response to many cancer chemotherapy drugs is even worse.
  • 5-fluorouracil is standard therapy for advanced colorectal cancer, but only about 20-40% of patients have an objective response to the drug, and, of these, only 1-5% of patients have a complete response (complete tumor disappearance; the remaining patients have only partial tumor shrinkage). Conversely, up to 20-30% of patients receiving 5-FU suffer serious gastrointestinal or hematopoietic toxicity, depending on the regimen.
  • the invention provides a method for selecting a treatment for a patient suffering from a disease or condition by determimng whether or not a gene or genes in cells of the patient (in some cases including both normal and disease cells, such as cancer cells) contain at least one sequence variance which is indicative of the effectiveness of the treatment of the disease or condition.
  • the gene is one specified herein, in particular one listed in a Table or list herein.
  • the at least one variance includes a plurality of variances which may provide a haplotype or haplotypes.
  • the joint presence of the plurality of variances is indicative of the potential effectiveness of the treatment in a patient having such plurality of variances.
  • the plurality of variances may each be indicative of the potential effectiveness of the treatment, and the effects of the individual variances may be independent or additive, or the plurality of variances may be indicative of the potential effectiveness if at least 2, 3, 4, or more appear jointly.
  • the plurality of variances may also be combinations of these relationships.
  • the plurality of variances may include variances from one, two, three or more gene loci.
  • the invention concerns a method for providing a correlation between a patient genotype and effectiveness of a treatment, by determining the presence or absence of a particular known variance or variances in cells of a patient for a gene of this invention, and providing a result indicating the expected effectiveness of a treatment for a disease or condition.
  • the result may be formulated by comparing the genotype of the patient with a list of variances indicative of the effectiveness of a treatment, e.g., administration of a drug described herein.
  • the determination may be by methods as described herein or other methods known to those skilled in the art.
  • the selection of a method of treatment may incorporate selection of one or more from a plurality of medical therapies.
  • the selection may be the selection of a method or methods which is/are more effective or less effective than certain other therapeutic regimens (with either having varying safety parameters).
  • the selection may be the selection of a method or methods which is safer than certain other methods of treatment in the patient.
  • the selection may involve either positive selection or negative selection or both, meaning that the selection can involve a choice that a particular method would be an appropriate method to use and/or a choice that a particular method would be an inappropriate method to use.
  • the presence of the at least one variance is indicative that the treatment will be effective or otherwise beneficial (or more likely to be beneficial) in the patient. Stating that the treatment will be effective means that the probability of beneficial therapeutic effect is greater than in a person not having the appropriate presence or absence of particular variances. In other embodiments, the presence of the at least one variance is indicative that the treatment will be ineffective or contra-indicated for the patient.
  • a treatment may be contra-indicated if the treatment results, or is more likely to result, in undesirable side effects, or an excessive level of undesirable side effects.
  • a determination of what constitutes excessive side-effects will vary, for example, depending on the disease or condition being treated, the availability of alternatives, the expected or experienced efficacy of the treatment, and the tolerance of the patient.
  • an effective treatment this means that it is more likely that a desired effect will result from the treatment administration in a patient with a particular variance or variances than in a patient who has a different variance or variances.
  • the presence of the at least one variance is indicative that the treatment is effective but results in undesirable effects or outcomes, e.g., has undesirable side-effects.
  • the term " tolerance” refers to the ability of a patient to accept a treatment, based, e.g., on deleterious effects and/or effects on lifestyle. Frequently, the term principally concerns the patients perceived magnitude of deleterious effects such as nausea, weakness, dizziness, and diarrhea, among others. Such experienced effects can, for example, be due to general or cell-specific toxicity, activity on non-target cells, cross-reactivity on non-target cellular constituents (non-mechanism based), and or side-effects of activity on the target cellular subsitutuent (mechanism based), or the cause of toxicity may not be understood. In any of these circumstances one may identify an association between the undesirable effects and variances in specific genes.
  • the variance or variant form or forms of a gene is/are associated with a specific response to a drug.
  • the frequency of a specific variance or variant form of the gene may correspond to the frequency of an efficacious response to administration of a drug.
  • the frequency of a specific variance or variant form of the gene may correspond to the frequency of an adverse event resulting from administration of a drug.
  • the frequency of a specific variance or variant form of a gene may not correspond closely with the frequency of a beneficial or adverse response, yet the variance may still be useful for identifying a patient subset with high response or toxicity incidence because the variance may account for only a fraction of the patients with high response or toxicity.
  • the drug will be effective in more than 20%) of individuals with one or more specific variances or variant forms of the gene, more preferably in 40% and most preferably in >60%.
  • the drug will be toxic or create clinically unacceptable side effects in more than 10% of individuals with one or more variances or variant forms of the gene, more preferably in >30%, more preferably in >50%, and most preferably in >70% or in more than 90%.
  • the method of selecting a treatment includes eliminating a treatment, where the presence or absence of the at least one variance is indicative that the treatment will be ineffective or contra-indicated.
  • the selection of a method of treatment can include identifying both a first and second treatment, where the first treatment is effective to treat the disease or condition, and the second treatment reduces a deleterious effect of the first treatment.
  • eliminating a treatment refers to removing a possible treatment from consideration, e.g., for use with a particular patient based on the presence or absence of a particular variance(s) in one or more genes in cells of that patient, or to stopping the administration of a treatment which was in the course of administration.
  • the treatment will involve the administration of a compound preferentially active in patients with a form or forms of a gene, where the gene is one identified herein.
  • the administration may involve a combination of compounds.
  • the method involves identifying such an active compound or combination of compounds, where the compound is less active or is less safe or both when administered to a patient having a different form of the gene.
  • the compound is a compound in a drug class identified in the 1999 Physicians' Desk Reference (53rd edition), Medical Economics Data, 1998, the PharmaProjects database, the IMS database or identified herein, e.g., in an exemplary drug table herein (see, e.g., Examples 6, 8, and 9 and Tables 7 and 9 herein).
  • the method of selecting a treatment involves selecting a method of administration of a compound, combination of compounds, or pharmaceutical composition, for example, selecting a suitable dosage level and/or frequency of administration, and/or mode of administration of a compound.
  • the method of administration can be selected to provide better, preferably maximum therapeutic benefit.
  • maximum refers to an approximate local maximum based on the parameters being considered, not an absolute maximum.
  • a "suitable dosage level” refers to a dosage level which provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Often this dosage level is related to the peak or catalog serum levels resulting from administration of a drug at the particular dosage level.
  • a “frequency of administration” refers to how often in a specified time period a treatment is administered, e.g., once, twice, or three times per day, every other day, once per week, etc.
  • the frequency of administration is generally selected to achieve a pharmacologically effective average or peak serum level without excessive deleterious effects (and preferably while still being able to have reasonable patient compliance for self-administered drugs).
  • a particular gene or genes can be relevant to more than one disease or condition, for example, the gene or genes can have a role in the initiation, development, course, treatment, treatment outcomes, or health-related quality of life outcomes of a number of different diseases, disorders, or conditions.
  • the disease or condition or treatment of the disease or condition is any which involves a particular gene.
  • the gene is a gene identified herein. Determining the presence of a particular variance or plurality of variances in a particular gene in a patient can be performed in a variety of ways.
  • the detection of the presence or absence of at least one variance involves amplifying a segment of nucleic acid including at least one of the at least one variances.
  • a segment of nucleic acid to be amplified is 500 nucleotides or less in length, more preferably 100 nucleotides or less, and most preferably 45 nucleotides or less.
  • the amplified segment or segments includes a plurality of variances, or a plurality of segments of a gene or of a plurality of genes.
  • determining the presence of a set of variances in a specific gene may entail a haplotyping test that requires allele-specific amplification of a large DNA segment of no greater than 20,000 nucleotides, preferably no greater than 10,000 nucleotides and more preferably no greater than 5,000 nucleotides.
  • one allele may be enriched by methods other than amplification prior to determining genotypes at specific variant positions on the enriched allele as a way of determining haplotypes.
  • the determination of the presence or absence of a variance involves determining the sequence of the variance site or sites by methods such as chain terminating DNA sequencing or minisequencing, or by oligonucleotide hybridization or by mass spectrometry.
  • gene in the context of this invention refers to the particular alleleic form of a gene, which can be defined by the particular nucleotide(s) present in a nucleic acid sequence at a particular site(s).
  • the detection of the presence or absence of the at least one variance involves contacting a nucleic acid sequence corresponding to one of the genes identified above or a product of such a gene with a probe.
  • the probe is able to distinguish a particular form of the gene or gene product or the presence or a particular variance or variances, e.g., by differential binding or hybridization.
  • exemplary probes include nucleic acid hybridization probes, peptide nucleic acid probes, nucleotide- containing probes which also contain at least one nucleotide analog, and antibodies, e.g., monoclonal antibodies, and other probes as discussed herein. Those skilled in the art are familiar with the preparation of probes with particular specificities.
  • determining the presence or absence of the at least one variance involves sequencing at least one nucleic acid sequence.
  • the sequencing involves sequencing of a portion or portions of a gene and/or portions of a plurality of genes which includes at least one variance site, and may include a plurality of such sites.
  • the portion is 500 nucleotides or less in length, more preferably 100 nucleotides or less, and most preferably 45 nucleotides or less in length.
  • Such sequencing can be carried out by various methods recognized by those skilled in the art, including use of dideoxy termination methods (e.g., using dye-labeled dideoxy nucleotides) and the use of mass spectrometric methods.
  • mass spectrometric methods may be used to determine the nucleotide present at a variance site.
  • the plurality of variances can constitute a haplotype or haplotypes.
  • variant form of a gene refers to one specific form of a gene in a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene.
  • sequences at these variant sites that differ between different alleles of the gene are termed "gene sequence variances" or "variances” or “variants”.
  • alternative form refers to an allele that can be distinguished from other alleles by having distinct variances at at least one, and frequently more than one, variant sites within the gene sequence.
  • Other terms known in the art to be equivalent include mutation and polymorphism, although mutation is often used to refer to an allele associated with a deleterious phenotype.
  • the variances are selected from the group consisting of the variances listed in the variance tables herein or in a patent or patent application referenced and incorporated by reference in this disclosure.
  • reference to the presence of a variance or variances means particular variances, i.e., particular nucleotides at particular polymorphic sites, rather than just the presence of any variance in the gene.
  • Variances occur in the human genome at approximately one in every 500 - 1,000 bases within the human genome when two alleles are compared. When multiple alleles from unrelated individuals are compared the frequency of variant sites increases. At most variant sites there are only two alternative nucleotides involving the substitution of one base for another or the insertion/deletion of one or more nucleotides. Within a gene there may be several variant sites. Variant forms of the gene or alternative alleles can be distinguished by the presence of alternative variances at a single variant site, or a combination of several different variances at different sites (haplotypes). It is estimated that there are 3,300,000,000 bases in the sequence of a single haploid human genome. All human cells except germ cells are normally diploid.
  • Each gene in the genome may span 100-10,000,000 bases of DNA sequence or 100-20,000 bases of mRNA. It is estimated that there are between 60,000 and 120,000 genes in the human genome.
  • the "identification" of genetic variances or variant forms of a gene involves the discovery of variances that are present in a population. The identification of variances is required for development of a diagnostic test to determine whether a patient has a variant form of a gene that is known to be associated with a disease, condition, or predisposition or with the efficacy or safety of the drug. Identification of previously undiscovered genetic variances is distinct from the process of "determimng" the status of known variances by a diagnostic test.
  • the present invention provides exemplary variances in genes listed in the gene tables, as well as methods for discovering additional variances in those genes and a comprehensive written description of such additional possible variances. Also described are methods for DNA diagnostic tests to determine the DNA sequence at a particular variant site or sites.
  • the process of "identifying" or discovering new variances involves comparing the sequence of at least two alleles of a gene, more preferably at least 10 alleles and most preferably at least 50 alleles, (keeping in mind that each somatic cell has two alleles).
  • the analysis of large numbers of individuals to discover variances in the gene sequence between individuals in a population will result in detection of a greater fraction of all the variances in the population.
  • the process of identifying reveals whether there is a variance within the gene; more preferably identifying reveals the location of the variance within the gene; more preferably identifying provides knowledge of the sequence of the nucleic acid sequence of the variance, and most preferably identifying provides knowledge of the combination of different variances that comprise specific variant forms of the gene or alleles.
  • identifying new variances it is often useful to screen different population groups based on racial, ethnic, gender, and/or geographic origin because particular variances may differ in frequency between such groups. It may also be useful to screen DNA from individuals with a particular disease or condition of interest because they may have a higher frequency of certain variances than the general population.
  • the process of determining involves using diagnostic tests for specific variances or variant forms of the gene (or genes) that have been identified within the gene. It will be apparent that such diagnostic tests can only be performed after variances and variant forms of the gene have been identified. Identification of variances can be performed by a variety of methods, alone or in combination, including, for example, DNA sequencing, SSCP, heteroduplex analysis, denaturing gradient gel electrophoresis (DGGE), heteroduplex cleavage (either enzymatic as with T4 Endonuclease 7, or chemical as with osmium tetroxide and hydroxylamine), computational methods (described herein), and other methods described herein as well as others known to those skilled in the art.
  • DGGE denaturing gradient gel electrophoresis
  • analyzing a sequence refers to determimng at least some sequence information about the sequence, e.g., determining the nucleotides present at particular sites in the sequence or determining the base sequence of all of a portion of the particular sequence.
  • haplotype refers to a cis arrangement of two or more polymorphic nucleotides, i.e., variances, on a particular chromosome, e.g., in a particular gene. The haplotype preserves the information of the phase of the polymorphic nucleotides - that is, which set of variances were inherited from one parent, and which from the other.
  • the frequency of the variance or variant form of the gene in a population is known.
  • Measures of frequency known in the art include "allele frequency", namely the fraction of genes in a population that have one specific variance or set of variances. The allele frequencies for any gene should sum to 1.
  • Another measure of frequency known in the art is the "heterozygote frequency” namely, the fraction of individuals in a population who carry two alleles, or two forms of a particular variance or variant form of a gene, one inherited from each parent.
  • the number of individuals who are homozygous for a particular form of a gene may be a useful measure.
  • the relationship between allele frequency, heterozygote frequency, and homozygote frequency is described for many genes by the Hardy- Weinberg equation, which provides the relationship between allele frequency, heterozygote frequency and homozygote frequency in a freely breeding population at equilibrium. Most human variances are substantially in Hardy- Weinberg equilibrium.
  • the allele frequency, heterozygote frequency, or homozygote frequency are determined experimentally.
  • a variance has an allele frequency of at least 0.01, more preferably at least 0.05, still more preferably at least 0.10.
  • the allele may have a frequency as low as 0.001 if the associated phenotype is a rare form of toxic reaction to the treatment or drug.
  • population refers to a geographically, ethnically, racially, gender, and/or culturally defined group of individuals or a group of individuals with a particular disease or condition or individuals that may be treated with a specific drug. In most cases a population will preferably encompass at least ten thousand, one hundred thousand, one million, ten million, or more individuals, with the larger numbers being more preferable. In a preferred aspect of this invention, the population refers to individuals with a specific disease or condition that may be treated with a specific drug. In an aspect of this invention, the allele frequency, heterozygote frequency, or homozygote frequency of a specific variance or variant form of a gene is known. In preferred embodiments of this invention, the frequency of one or more variances that may predict response to a treatment is determined in one or more populations using a diagnostic test.
  • probe refers to a molecule which can detectably distinguish between target molecules differing in structure. Detection can be accomplished in a variety of different ways depending on the type of probe used and the type of target molecule. Thus, for example, detection may be based on discrimination of activity levels of the target molecule, but preferably is based on detection of specific binding. Examples of such specific binding include antibody binding and nucleic acid probe hybridization. Thus, for example, probes can include enzyme substrates, antibodies and antibody fragments, and nucleic acid hybridization probes.
  • the detection of the presence or absence of the at least one variance involves contacting a nucleic acid sequence which includes a variance site with a probe, preferably a nucleic acid probe, where the probe preferentially hybridizes with a form of the nucleic acid sequence containing a complementary base at the variance site as compared to hybridization to a form of the nucleic acid sequence having a non- complementary base at the variance site, where the hybridization is carried out under selective hybridization conditions.
  • a nucleic acid hybridization probe may span two or more variance sites.
  • a nucleic acid probe can include one or more nucleic acid analogs, labels or other substituents or moieties so long as the base- pairing function is retained.
  • administration of a particular treatment e.g., administration of a therapeutic compound or combination of compounds
  • the disease or condition is one for which administration of a treatment is expected to provide a therapeutic benefit
  • the compound is a compound identified herein, e.g., in a drug table such as Tables 7 and 9.
  • the terms “effective” and “effectiveness” includes both pharmacological effectiveness and physiological safety.
  • Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient.
  • Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment.
  • side-effects the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment.
  • the term “ineffective” indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the total (unstratified) population.
  • Treatment may be effective in a subgroup that can be identified by the presence of one or more sequence variances or alleles.
  • Less effective means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects.
  • a drug which is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.
  • deleterious effects refers to physical effects in a patient caused by administration of a treatment which are regarded as medically undesirable.
  • deleterious effects can include a wide spectrum of toxic effects injurious to health such as death of normal cells when only death of diseased cells is desired, nausea, fever, inability to retain food, dehydration, damage to critical organs such as renal tubular necrosis, fatty liver or pulmonary fibrosis, among many others.
  • contra-indicated means that a treatment results in deleterious effects such that a prudent medical doctor treating such a patient would regard the treatment as unsuitable for administration.
  • the variance information is used to select both a first method of treatment and a second method of treatment.
  • the first treatment is a primary treatment which provides a physiological effect directed against the disease or condition or its symptoms.
  • the second method is directed to reducing or eliminating one or more deleterious effects of the first treatment, e.g., to reduce a general toxicity or to reduce a side effect of the primary treatment.
  • the second method can be used to allow use of a greater dose or duration of the first treatment, or to allow use of the first treatment in patients for whom the first treatment would not be tolerated or would be contra-indicated in the absence of a second method to reduce deleterious effects.
  • the invention provides a method for selecting a method of treatment for a patient suffering from a disease or condition by comparing at least one variance in at least one gene in the patient, with a list of variances in the gene or genes which are indicative of the effectiveness of at least one method of treatment.
  • the comparison involves a plurality of variances or a haplotype indicative of the effectiveness of at least one method of treatment.
  • the list of variances includes a plurality of variances.
  • the at least one method of treatment involves the administration of a compound effective in at least some patients with a disease or condition; the presence or absence of the at least one variance is indicative that the treatment will be effective in the patient; and/or the presence or absence of the at least one variance is indicative that the treatment will be ineffective or contra-indicated in the patient; and/or the treatment is a first treatment and the presence or absence of the at least one variance is indicative that a second treatment will be beneficial to reduce a deleterious effect of the first treatment; and/or the at least one treatment is a plurality of methods of treatment.
  • the selecting involves determining whether any of the methods of treatment will be more effective than at least one other of the plurality of methods of treatment.
  • Yet other embodiments are provided as described for the preceding aspect in connection with methods of treatment using administration of a compound; treatment of various diseases, and variances in particular genes.
  • the term "list” refers to one or more variances which have been identified for a series or genes of potential importance in accounting for inter-individual variation in treatment response.
  • the list is recorded in written or electronic form.
  • variances are recorded in Tables 3, 4, and 10 and additional gene variance identification tables herein in a form which allows comparison with other variance information.
  • the invention also provides a method for selecting a method of administration of a compound to a patient suffering from a disease or condition, by determining the presence or absence of at least one variance in cells of the patient in a gene which is a gene selected from the genes identified in a gene table or list below, where such presence or absence is indicative of an appropriate method of administration of the compound.
  • the selection of a method of treatment involves selecting a dosage level or frequency of administration or route of administration of the compound or combinations of those parameters.
  • two or more compounds are to be administered, and the selecting involves selecting a method of administration for one, two, or more than two of the compounds, jointly, concurrently, or separately.
  • selecting a method of administration for one, two, or more than two of the compounds jointly, concurrently, or separately.
  • such plurality of compounds is often used in combination therapy, and thus may be formulated in a single drug, or may be separate drugs administered concurrently, serially, or separately.
  • Other embodiments are as indicated above for selection of second treatment methods, methods of identifying variances, and methods of treatment as described for aspects above.
  • the invention provides a method for selecting a patient for administration of a method of treatment for a disease or condition, or of selecting a patient for a method of administration of a treatment, by comparing the presence or absence of at least one variance in a gene as identified above in cells of a patient, with a list of variances in the gene, where the presence or absence of the at least one variance is indicative that the treatment or method of administration will be effective in the patient. If the at least one variance is present in the patient's cells, then the patient is selected for administration of the treatment.
  • the disease or the method of treatment is as described in aspects above, specifically including, for example, those described for selecting a method of treatment
  • the invention provides a method for identifying a subset of patients with enhanced or diminished response or tolerance to a treatment method or a method of admimstration of a treatment where the treatment is for a disease or condition in the patient.
  • the method involves correlating one or more variances in one or more genes in a plurality of patients with response to a treatment or a method of administration of a treatment.
  • the co ⁇ elation may be performed by determining the one or more variances in the one or more genes in the plurality of patients and correlating the presence or absence of each of the variances (alone or in various combinations) with the patient's response to treatment.
  • the variances may be previously known to exist or may also be determined in the present method or combinations of prior information and newly determined information may be used.
  • a positive correlation between the presence of one or more variances and an enhanced response to treatment is indicative that the treatment is particularly effective in the group of patients having those variances.
  • a positive co ⁇ elation of the presence of the one or more variances with a diminished response to the treatment is indicative that the treatment will be less effective in the group of patients having those variances.
  • Such information is useful, for example, for selecting or deselecting patients for a particular treatment or method of admimstration of a treatment, or for demonstrating that a group of patients exists for which the treatment or method of treatment would be particularly beneficial or contra-indicated.
  • Such demonstration can be beneficial, for example, for obtaining government regulatory approval for a new drug or a new use of a drug.
  • the variances are in particular genes, or are particular variances described herein. Also, preferred embodiments include drugs, treatments, variance identification or determination, determination of effectiveness, lists, and/or diseases as described for aspects above or otherwise described herein.
  • the correlation of patient responses to therapy according to patient genotype is carried out in a clinical trial, e.g., as described herein according to any of the variations described..
  • Detailed description of methods for associating variances with clinical outcomes using clinical trials are provided below.
  • the selection may be positive selection or negative selection.
  • the methods can include eliminating a treatment for a patient, eliminating a method or mode of administration of a treatment to a patient, or elimination of a patient for a treatment or method of treatment.
  • the methods can involve such identification or comparison for a plurality of genes.
  • the genes are functionally related to the same disease or condition, or to the aspect of disease pathophysiology that is being subjected to pharmacological manipulation by the treatment (e.g. a drug), or to the activation or inactivation of the drug, and more preferably the genes are involved in the same biochemical process or pathway.
  • the invention provides a method for identifying the forms of a gene in an individual, where the gene is one specified as for aspects above, by determining the presence or absence of at least one variance in the gene.
  • the at least one variance includes at least one variance selected from the group of variances identified in variance tables herein.
  • the presence or absence of the at least one variance is indicative of the effectiveness of a therapeutic treatment in a patient suffering from a disease or condition and having cells containing the at least one variance.
  • the presence or absence of the variances can be determined in any of a variety of ways as recognized by those skilled in the art.
  • the nucleotide sequence of at least one nucleic acid sequence which includes at least one variance site can be determined, such as by chain termination methods, hybridization methods or by mass spectrometric methods.
  • the determining involves contacting a nucleic acid sequence or a gene product of one of one of the genes with a probe which specifically identifies the presence or absence of a form of the gene.
  • a probe e.g., a nucleic acid probe
  • a probe which specifically binds, e.g., hybridizes, to a nucleic acid sequence corresponding to a portion of the gene and which includes at least one variance site under selective binding conditions.
  • determining the presence or absence of at least two variances can constitute determimng a haplotype or haplotypes.
  • variances related to types of treatment include variances related to types of treatment, drug responses, diseases, nucleic acid sequences, and other items related to variances and variance determination as described for aspects above.
  • the invention provides a pharmaceutical composition which includes a compound which has a differential effect in patients having at least one copy, or alternatively, two copies of a form of a gene as identified for aspects above and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the composition is adapted to be preferentially effective to treat a patient with cells containing the one, two, or more copies of the form of the gene.
  • the material is subject to a regulatory limitation or restriction on approved uses or indications, e.g., by the U.S. Food and Drug Administration (FDA), limiting approved use of the composition to patients having at least one copy of the particular form of the gene which contains at least one variance.
  • the composition is subject to a regulatory limitation or restriction on approved uses indicating that the composition is not approved for use or should not be used in patients having at least one copy of a form of the gene including at least one variance.
  • the composition is packaged, and the packaging includes a label or insert indicating or suggesting beneficial therapeutic approved use of the composition in patients having one or two copies of a form of the gene including at least one variance.
  • the label or insert limits approved use of the composition to patients having zero or one or two copies of a form of the gene including at least one variance.
  • the latter embodiment would be likely where the presence of the at least one variance in one or two copies in cells of a patient means that the composition would be ineffective or deleterious to the patient.
  • the composition is indicated for use in treatment of a disease or condition which is one of those identified for aspects above.
  • the at least one variance includes at least one variance from those identified herein.
  • packaged means that the drug, compound, or composition is prepared in a manner suitable for distribution or shipping with a box, vial, pouch, bubble pack, or other protective container, which may also be used in combination.
  • the packaging may have printing on it and/or printed material may be included in the packaging.
  • the drug is selected from the drug classes or specific exemplary drugs identified in an example, in a table or list herein, and is subject to a regulatory limitation or suggestion or warning as described above that limits or suggests limiting approved use to patients having specific variances or variant forms of a gene identified in Examples or in a gene list provided below in order to achieve maximal benefit and avoid toxicity or other deleterious effect.
  • a pharmaceutical composition can be adapted to be preferentially effective in a variety of ways.
  • an active compound is selected which was not previously known to be differentially active, or which was not previously recognized as a potential therapeutic compound.
  • the concentration of an active compound which has differential activity can be adjusted such that the composition is appropriate for administration to a patient with the specified variances. For example, the presence of a specified variance may allow or require the administration of a much larger dose, which would not be practical with a previously utilized composition. Conversely, a patient may require a much lower dose, such that administration of such a dose with a prior composition would be impractical or inaccurate.
  • the composition may be prepared in a higher or lower unit dose form, or prepared in a higher or lower concentration of the active compound or compounds.
  • the composition can include additional compounds needed to enable administration of a particular active compound in a patient with the specified variances, which was not in previous compositions, e.g., because the majority of patients did not require or benefit from the added component.
  • the term “differential” or “differentially” generally refers to a statistically significant different level in the specified property or effect. Perferably, the difference is also functionally significant.
  • “differential binding or hybridization” is sufficient difference in binding or hybridization to allow discrimination using an appropriate detection technique.
  • “differential effect” or “differentially active” in connection with a therapeutic treatment or drug refers to a difference in the level of the effect or activity which is distinguishable using relevant parameters and techniques for the effect or activity being considered.
  • the difference in effect or activity is also sufficient to be clinically significant, such that a co ⁇ esponding difference in the course of treatment or treatment outcome would be expected, at least on a probabilistic basis.
  • probes which specifically recognize a nucleic acid sequence co ⁇ esponding to a variance or variances in a gene or a product expressed from the gene, and are able to distinguish a variant form of the sequence or gene or gene product from one or more other variant forms of that sequence, gene, or gene product under selective conditions.
  • Those skilled in the art recognize and understand the identification or determination of selective conditions for particular probes or types of probes.
  • An exemplary type of probe is a nucleic acid hybridization probe, which will selectively bind under selective binding conditions to a nucleic acid sequence or a gene product co ⁇ esponding to one or the genes identified for aspects above.
  • probe is a peptide or protein, e.g., an antibody or antibody fragment which specifically or preferentially binds to a polypeptide expressed from a particular form of a gene as characterized by the presence or absence of at least one variance.
  • a "probe” is a molecule, commonly a nucleic acid, though also potentially a protein, carbohydrate, polymer, or small molecule, that is capable of binding to one variance or variant form of the gene or gene product to a greater extent than to a form of the gene having a different base at one or more variance sites, such that the presence of the variance or variant form of the gene can be determined.
  • the probe distinguishes at least one variance identified in Examples, tables or lists below.
  • the probe also has specificity for the particular gene or gene product, at least to an extent such that binding to other genes or gene products does not prevent use of the assay to identify the presence or absence of the particular variance or variances of interest.
  • the probe is an antibody or antibody fragment. Such antibodies may be polyclonal or monoclonal antibodies, and can be prepared by methods well-known in the art.
  • the probe is a nucleic acid probe at least 15, preferably at least 17 nucleotides in length, more preferably at least 20 or 22 or 25, preferably 500 or fewer nucleotides in length, more preferably 200 or 100 or fewer, still more preferably 50 or fewer, and most preferably 30 or fewer.
  • the probe has a length in a range between from any one of the above lengths to any other of the above lengths (including endpoints).
  • the probe specifically hybridizes under selective hybridization conditions to a nucleic acid sequence co ⁇ esponding to a portion of one of the genes identified in connection with above aspects.
  • the nucleic acid sequence includes at least one and preferably two or more variance sites.
  • the probe has a detectable label, preferably a fluorescent label. A variety of other detectable labels are known to those skilled in the art.
  • Such a nucleic acid probe can also include one or more nucleic acid analogs.
  • the probe is an antibody or antibody fragment which specifically binds to a gene product expressed from a form of one of the above genes, where the form of the gene has at least one specific variance with a particular base at the variance site, and preferably a plurality of such variances.
  • the term “ specifically hybridizes” indicates that the probe hybridizes to a sufficiently greater degree to the target sequence than to a sequence having a mismatched base at at least one variance site to allow distinguishing such hybridization.
  • the term “ specifically hybridizes” thus means that the probe hybridizes to the target sequence, and not to non-target sequences, at a level which allows ready identification of probe/target sequence hybridization under selective hybridization conditions.
  • selective hybridization conditions refer to conditions which allow such differential binding.
  • the terms “ specifically binds” and “selective binding conditions” refer to such differential binding of any type of probe, e.g., antibody probes, and to the conditions which allow such differential binding.
  • hybridization reactions to determine the status of variant sites in patient samples are carried out with two different probes, one specific for each of the (usually two) possible variant nucleotides.
  • the complementary information derived from the two separate hybridization reactions is useful in co ⁇ oborating the results.
  • the invention provides an isolated, purified or enriched nucleic acid sequence of 15 to 500 nucleotides in length, preferably 15 to 100 nucleotides in length, more preferably 15 to 50 nucleotides in length, and most preferably 15 to 30 nucleotides in length, which has a sequence which co ⁇ esponds to a portion of one of the genes identified for aspects above.
  • the lower limit for the preceding ranges is 17, 20, 22, or 25 nucleotides in length.
  • the nucleic acid sequence is 30 to 300 nucleotides in length, or 45 to 200 nucleotides in length, or 45 to 100 nucleotides in length.
  • the nucleic acid sequence includes at least one variance site.
  • sequences can, for example, be amplification products of a sequence which spans or includes a variance site in a gene identified herein.
  • a sequence can be a primer which is able to bind to or extend through a variance site in such a gene.
  • a nucleic acid hybridization probe comprised of such a sequence.
  • the nucleotide sequence can contain a sequence or site co ⁇ esponding to a variance site or sites, for example, a variance site identified herein.
  • the presence or absence of a particular variant form in the heterozygous or homozygous state is indicative of the effectiveness of a method of treatment in a patient.
  • primers are utilized in pairs.
  • Primers can be designed or selected by methods well-known to those skilled in the art based on nucleotide sequences co ⁇ esponding to at least a portion or a gene identified herein.
  • the primer or primers hybridizes to or allows amplification (e.g., using the polymerase chain reaction) through a nucleic acid sequence containing at least one sequence variance.
  • primers hybridize to a sequence not more than 300 nucleotides, more preferably not more than 200 nucleotides, still more preferably not more than 100 nucleotides, and most preferably not more than 50 nucleotides away from a variance site which is to be analyzed.
  • a primer is 100 nucleotides or fewer in length, more preferably 50 nucleotides or fewer, still more preferable 30 nucleotides or fewer, and most preferably 20 or fewer nucleotides in length.
  • the te ⁇ n " co ⁇ espond” refers to a nucleotide sequence relationship, such that the nucleotide sequence has a nucleotide sequence which is the same as the reference gene or an indicated portion thereof, or has a nucleotide sequence which is exactly complementary in normal Watson-Crick base pairing, or is an RNA equivalent of such a sequence, e.g., a mRNA, or is a cDNA derived from an mRNA of the gene.
  • the invention provides a kit containing at least one probe or at least one primer or both (e.g., as described above) co ⁇ esponding to a gene or genes of this invention.
  • the kit is preferably adapted and configured to be suitable for identification of the presence or absence of a particular variance or variances, which can include or consist of sequence a nucleic acid sequence co ⁇ esponding to a portion of a gene.
  • the kit may also contain a plurality of either or both of such probes and/or primers, e.g., 2, 3, 4, 5, 6, or more of such probes and/or primers.
  • the plurality of probes and/or primers are adapted to provide detection of a plurality of different sequence variances in a gene or plurality of genes, e.g., in 2, 3, 4, 5, or more genes or to sequence a nucleic acid sequence including at least one variance site in a gene or genes.
  • one or more of the variance or variances to be detected are co ⁇ elated with variability in a treatment response or tolerance, and are preferably indicative of an effective response to a treatment.
  • the kit contains components (e.g., probes and/or primers) adapted or useful for detection of a plurality of variances (which may be in one or more genes) indicative of the effectiveness of at least one treatment, preferably of a plurality of different treatments for a particular disease or condition. It may also be desirable to provide a kit containing components adapted or useful to allow detection of a plurality of variances indicative of the effectiveness of a treatment or treatment against a plurality of diseases. The kit may also optionally contain other components, preferably other components adapted for identifying the presence of a particular variance or variances.
  • Such additional components can, for example, independently include a buffer or buffers, e.g., amplification buffers and hybridization buffers, which may be in liquid or dry form, a DNA polymerase, e.g., a polymerase suitable for carrying out PCR, and deoxy nucleotide triphosphases (dNTPs).
  • a probe includes a detectable label, e.g., a fluorescent label, enzyme label, light scattering label, or other label.
  • the kit includes a nucleic acid or polypeptide a ⁇ ay.
  • the a ⁇ ay may, for example, include a plurality of different antibodies, a plurality of different nucleic acid sequences.
  • Sites in the a ⁇ ay can allow capture and/or detection of nucleic acid sequences or gene products co ⁇ esponding to different variances in one or more different genes.
  • the a ⁇ ay is a ⁇ anged to provide variance detection for a plurality of variances in one or more genes which co ⁇ elate with the effectiveness of one or more treatments of one or more diseases.
  • the kit may also optionally contain instructions for use, which can include a listing of the variances co ⁇ elating with a particular treatment or treatments for a disease of diseases.
  • the kit components are selected to allow detection of a variance described herein, and/or detection of a variance indicative of a treatment,e.g., administration of a drug, pointed out herein. Additional configurations for kits of this invention will be apparent to those skilled in the art.
  • the invention provides a method for determining a genotype of an individual in relation to one or more variances in one or more of the genes identified in above aspects by using mass spectrometric determination of a nucleic acid sequence which is a portion of a gene identified for other aspects of this invention or a complementary sequence.
  • mass spectrometric methods are known to those skilled in the art.
  • the method involves determining the presence or absence of a variance in a gene; determining the nucleotide sequence of the nucleic acid sequence; the nucleotide sequence is 100 nucleotides or less in length, preferably 50 or less, more preferably 30 or less, and still more preferably 20 nucleotides or less.
  • such a nucleotide sequence includes at least one variance site, preferably a variance site which is informative with respect to the expected response of a patient to a treatment as described for above aspects.
  • the invention provides a method for determining whether a compound has a differential effect due to the presence or absence of at least one variance in a gene or a variant form of a gene, where the gene is a gene identified for aspects above.
  • the method involves identifying a first patient or set of patients suffering from a disease or condition whose response to a treatment differs from the response (to the same treatment) of a second patient or set of patients suffering from the same disease or condition, and then determining whether the frequency of at least one variance in at least one gene differs in frequency between the first patient or set of patients and the second patient or set of patients.
  • a co ⁇ elation between the presence or absence of the variance or variances and the response of the patient or patients to the treatment indicates that the variance provides information about variable patient response.
  • the method will involve identifying at least one variance in at least one gene.
  • An alternative approach is to identify a first patient or set of patients suffering from a disease or condition and having a particular genotype, haplotype or combination of genotypes or haplotypes, and a second patient or set of patients suffering from the same disease or condition that have a genotype or haplotype or sets of genotypes or haplotypes that differ in a specific way from those of the first set of patients. Subsequently the extent and magnitude of clinical response can be compared between the first patient or set of patients and the second patient or set of patients. A co ⁇ elation between the presence or absence of a variance or variances or haplotypes and the response of the patient or patients to the treatment indicates that the variance provides information about variable patient response and is useful for the present invention.
  • the method can utilize a variety of different informative comparisons to identify co ⁇ elations. For example a plurality of pairwise comparisons of treatment response and the presence or absence of at least one variance can be performed for a plurality of patients. Likewise, the method can involve comparing the response of at least one patient homozygous for at least one variance with at least one patient homozygous for the alternative form of that variance or variances. The method can also involve comparing the response of at least one patient heterozygous for at least one variance with the response of at least one patient homozygous for the at least one variance.
  • heterozygous patient response is compared to both alternative homozygous fo ⁇ ns, or the response of heterozygous patients is grouped with the response of one class of homozygous patients and said group is compared to the response of the alternative homozygous group.
  • Such methods can utilize either retrospective or prospective information concerning treatment response variability.
  • patient response to the method of treatment is variable.
  • the disease or condition is as for other aspects of this invention; for example, the treatment involves administration of a compound or pharmaceutical composition.
  • the method involves a clinical trial, e.g., as described herein.
  • a trial can be a ⁇ anged, for example, in any of the ways described herein, e.g., in the Detailed Description.
  • the present invention also provides methods of treatment of a disease or condition. Such methods combine identification of the presence or absence of particular variances with the administration of a compound; identification of the presence of particular variances with selection of a method of treatment and administration of the treatment; and identification of the presence or absence of particular variances with elimination of a method of treatment based on the variance information indicating that the treatment is likely to be ineffective or contra-indicated, and thus selecting and administering an alternative treatment effective against the disease or condition.
  • prefe ⁇ ed embodiments of these methods incorporate prefe ⁇ ed embodiments of such methods as described for such sub-aspects.
  • a “gene” is a sequence of DNA present in a cell that directs the expression of a "biologically active” molecule or “gene product”, most commonly by transcription to produce RNA and translation to produce protein.
  • the "gene product 1 is most commonly a RNA molecule or protein or a RNA or protein that is subsequently modified by reacting with, or combining with, other constituents of the cell. Such modifications may include, without limitation, modification of proteins to form glycoproteins, Hpoproteins, and phosphoproteins, or other modifications known in the art.
  • RNA may be modified without limitation by complexing with proteins, polyadenylation, splicing, capping or export from the nucleus.
  • gene product refers to any product directly resulting from transcription of a gene.
  • this includes partial, precursor, and mature transcription products (i.e, pre-mRNA and mRNA), and translation products with or without further processing including, without limitation, lipidation, phosphorylation, glycosylation, or combinations of such processing
  • gene involved in the origin or pathogenesis of a disease or condition refers to a gene that harbors mutations that contribute to the cause of disease, or variances that affect the progression of the disease or expression of specific characteristic of the disease.
  • the term also applies to genes involved in the synthesis, accumulation, or elimination of products that are involved in the origin or pathogenesis of a disease or condition including, without limitation, proteins, lipids, carbohydrates, hormones, or small molecules.
  • gene involved in the action of a drug refers to any gene whose gene product affects the efficacy or safety of the drug or affects the disease process being treated by the drug, and includes, without limitation, genes that encode gene products that are targets for drug action, gene products that are involved in the metabolism, activation or degradation of the drug, gene products that are involved in the bioavailability or elimination of the drug to the target, gene products that affect biological pathways that, in rum, affect the action of the drug such as the synthesis or degradation of competitive substrates or allosteric effectors or rate limiting reaction, or, alternatively, gene products that affect the pathophysiology of the disease process.
  • Particular variances in the latter category of genes may be associated with patient groups in whom disease etiology is more or less susceptible to amelioration by the drug. For example, there are several pathophysiological mechanisms in hypertension, and depending on the dominant mechanism in a given patient, that patient may be more or less likely than the average hypertensive patient to respond to a drug that primarily targets one pathophysiological mechanism. The relative importance of different pathophysiological mechanisms in individual patients is likely to be affected by variances in genes associated with the disease pathophysiology.
  • the "action" of a drug refers to its effect on biological products within the body.
  • the action of a drug also refers to its effects on the signs or symptoms of a disease or condition, or effects of the drug that are unrelated to the disease or condition leading to unanticipated effects on other processes. Such unanticipated processes often lead to adverse events or toxic effects.
  • adverse event or "toxic” event” are known in the art and include, without limitation, those listed in the FDA reference system for adverse events.
  • drugs that are explicitly indicated for, and/or for which approved use is restricted to individuals in the population with specific variances or combinations of variances, as determined by diagnostic tests for variances or variant forms of certain genes involved in the disease or condition or involved in the action of the drug.
  • Such drugs may provide more effective treatment for a disease or condition in a population identified or characterized with the use of a diagnostic test for a specific variance or variant form of the gene if the gene is involved in the action of the drug or in determining a characteristic of the disease or condition.
  • Such drugs may be developed using the diagnostic tests for specific variances or variant forms of a gene to determine the inclusion of patients in a clinical trial.
  • the invention also provides a method for producing a pharmaceutical composition by identifying a compound which has differential activity against a disease or condition in patients having at least one va ⁇ ance in a gene, compounding the pharmaceutical composition by combining the compound with a pharmaceutically acceptable earner, excipient. or diluent such that the composition is preferentially effective in patients who have at least one copy of the va ⁇ ance or vanances. In some cases, the patient has two copies of the va ⁇ ance or va ⁇ ances.
  • the disease or condition, gene or genes, vanances, methods of administration, or method of determimng the presence or absence of vanances is as descnbed for other aspects of this invention
  • the invention provides a method forproducing a pharmaceutical agent by identifying a compound which has differential activity against a disease or condition in patients having at least one copy of a form of a gene having at least one vanance and synthesizing the compound in an amount sufficient to provide a pharmaceutical effect in a patient suffenng from the disease or condition
  • the compound can be identified by conventional screening methods and its activity confirmed.
  • compound hbranes can be screened to identify compounds which differentially bind to products of va ⁇ ant forms of a particular gene product, or which differentially affect expression of va ⁇ ant forms of the particular gene, or which differentially affect the activity of a product expressed from such gene
  • Prefe ⁇ ed embodiments are as for the preceding aspect.
  • the invention provides a method of treating a disease or condition in a patient by selecting a patient whose cells have an allele of a gene selected from the genes listed herein, preferably in Tables 2, 6, or 8
  • the allele contains at least one vanance co ⁇ elated with more effective response to a treatment of the disease or condition, or tolerance of a treatment, e g , a treatment with a drug or a drug of a class indicated herein
  • the allele contains a vanance as shown in Tables 2, 6, or 8 or other vanance table herein
  • the alternateng involves admimstenng to the patient a compound preferentially active on at least one but less than all alleles of the gene.
  • the invention provides a method for determining a method of treatment effective to treat a disease or condition by alternativeng the level of activity of a product of an allele of a gene selected from the genes listed in Table 2, 6, or 8, and determining whether that alteration provides a differential effect related to reducing or alleviating a disease or condition as compared to at least one alternative allele or an alteration in toxicity or tolerance of the treatment by a patient or patients.
  • the presence of such a differential effect indicates that altering that level of activity provides at least part of an effective treatment for the disease or condition.
  • the determining is carried out in a clinical trial, e.g., as described above and/or in the Detailed Description below.
  • the invention provides a method for evaluating differential efficacy of or tolerance to a treatment in a subset of patients who have a particular variance or variances in at least one gene by utilizing a clinical trial.
  • the clinical trial is a Phase I, II, III, or IV trial.
  • Prefe ⁇ ed embodiments include the stratifications and/or analyses as described below in the Detailed Description.
  • the invention provides a method for identifying at least one variance in at least one gene using computer-based sequence analysis or variance scanning as known to those skilled in the art.
  • the at least one gene is a plurality of genes, preferably at least 10, 20,
  • sequence and/or variance information on the plurality of genes is acumulated in one database or a set of commonly accessible databases within a single local computer network or on a single computer.
  • the invention provides experimental methods for finding additional variances in any of the genes provided in the table of Table 2, 6, or 8.
  • sequence analysis method a number of experimental methods can also beneficially be used to identify variances.
  • the invention provides methods for producing cDNA (e.g., example 13) or genomic DNA and detecting additional variances in the genes provided in Table 2, 6, or 8 using the single strand conformation polymo ⁇ hism (SSCP) method (Example 14), the T4 Endonuclease VII method (Example 15) or DNA sequencing (Example 16) or other methods pointed out below.
  • SSCP single strand conformation polymo ⁇ hism
  • T4 Endonuclease VII method Example 15
  • DNA sequencing Example 16
  • the method preferably involves determining the presence or absence using a cell sample from an individual or individuals.
  • the methods can also involve obtaining a cell sample from an individual.
  • the cell sample can be any of a variety of different cells, e.g., blood cells skin cells, muscle cells, normal cells, or cancer cells.
  • FIG. 1 is a diagram showing the relationships of enzymes involved in 5- FU metabolism and inhibition of thymidylate formation. Enzymes: 1. uridine phosphorylase; 2. thymidine phosphorylase; 3. orotate phosphoribosyl transferase; 4. thymidine kinase; 5. uridine kinase; 6. ribonucletide reductase; 7. thymidylate synthase; 8. dCMP deaminase; 9. nucleoside monophosphate kinase; 10. nucleoside diphosphate kinase; 11.
  • FH2 dihydrofolate
  • FH4 tetrahydrofolate. The Figure is adapted from Goodman & Gilman's The Pharmacological Basis of Therapeutics, ninth edition, McGraw Hill, 1996, p. 1249.
  • FIG. 2 is a diagram showing the relationship of enzymes related to folate metabolism and formation of 5, 10-methylenetetrahydro folate.
  • Enzymes 1. Forminino-tetrahydro folate cyclodeaminase; 2. methenyltetrahydro folate synthetase; 3. methenyltetra-hydro folate cyclohydrolase; 4. formyltetrahydro folate synthetase; 5. formyltetrahydro folate hydrolase; 6. formyltetrahydro folate dehydrogenase; 7. methyleneltetrahydro folate dehydrogenase; 8. methyleneltetrahydrofolate reductase (MTHFR); 9.
  • homocysteine methyltransferase also called methionine synthetase
  • 10. serine transhydroxymethylase 1 1. glycine cleavage system; 12. thymidylate synthase; 13. dihydrofolate reductase.
  • THF tetrahydrofolate
  • DHF dihydrofolate. Note that THF appears twice (i.e. the product of step 6 is also substrate for enzymes 10 and 11. Step 12 also appears in Figure 1, above. This Figure is adapted from Mathews & van Holde, Biochemistry, The Benjamin/Cummings Publishing Co., Redwood City CA, 1990, page 697.
  • the present invention is generally described below in connection with cancer chemotherapy. However, the described approach and techniques are applicable to a variety of other treatments and to genes associated with the efficacy and safety of such other treatments, for example, genes function in the pathways identified below, along with the specific genes listed.
  • the present invention identifies a number of genes in certain treatment-related pathways, and further identifies a number of genetic sequence variances in those genes.
  • the present description further describes how to identify variances which co ⁇ elate with variable treatment efficacy and further how to identify additional variances in the identified genes and how to determine the treatment response co ⁇ elation of those additional variances.
  • Chemotherapy of cancer currently involves use of highly toxic drugs with na ⁇ ow therapeutic indices. Although progress has been made in the chemotherapeutic treatment of selected malignancies, most adult solid cancers remain highly refractory to treatment. Nonetheless, chemotherapy is the standard of care for most disseminated solid cancers. Chemotherapy often results in a significant fraction of treated patients suffering unpleasant or life-threatening side effects while receiving little or no clinical benefit; other patients may suffer few side effects and/or have complete remission or even cure. Any test that could predict response to chemotherapy, even partially, would allow more selective use of toxic drugs, and could thereby significantly improve efficacy of oncologic drug use, with the potential to both reduce side effects and increase the fraction of responders.
  • Chemotherapy is also expensive, not just because the drugs are often costly, but also because a ⁇ riimstering highly toxic drugs requires close monitoring by carefully trained personnel, and because hospitalization is often required for treatment of (or monitoring for) toxic drug reactions. Information that would allow patients to be divided into likely responder vs. non-responder (or likely side effect) groups, with only the former to receive treatment, would therefore also have a significant impact on the economics of cancer drug use.
  • Advances can be implemented by aiding identification of genetic markers associated with inte ⁇ atient variation in response during preclinical development (thereby allowing development of non-allele selective agents), or by identification or optimization of clinical trial design parameters in order to achieve successful development of therapeutic products at any stage of clinical development, or by identifying variables that will allow safe and efficacious use of a marketed product. Such advances will provide benefits in the form of therapeutic alternatives to those patients in need of medical care.
  • the processes are as follows: a) variability between patients in the response to a particular treatment is observed; b) at least a portion of the variable response is co ⁇ elated with the presence or absence of at least one variance in at least one gene; c) an analytical or diagnostic test is provided to determine the presence or absence of the at least one variance in individual patients; d) the presence or absence of the variance or variances is used to select a patient for a treatment or to select a treatment for a patient, or the variance information is used in other methods described herein.
  • Inte ⁇ atient variability is the rule, not the exception, in clinical therapeutics.
  • One of the best sources of information on inte ⁇ atient variability is the nurses and physicians supervising the clinical trial who accumulate a body of first hand observations of physiological responses to the drug in different normal subjects or patients.
  • Evidence of inte ⁇ atient variation in response can also be measured statistically, and may be best described by statistical measures that examine magnitude of response (beneficial or adverse) across a large number of subjects.
  • the present invention concerns DNA sequence variances that can affect one or more of: i. The susceptibility of individuals to a disease; ii. The course or natural history of a disease; iii. The response of a patient with a disease to a medical intervention, such as, for example, a drug, a biologic substance, physical energy such as radiation therapy, or a specific dietary regimen. The ability to predict either beneficial or detrimental responses is medically useful. Thus variation in any of these three parameters may constitute the basis for initiating a pharmacogenetic study directed to the identification of the genetic sources of inte ⁇ atient variation.
  • the effect of a DNA sequence variance or variances on disease susceptibility or natural history are of particular interest as the variances can be used to define patient subsets which behave differently in response to medical interventions such as those described in (iii).
  • a variance can be useful for customizing medical therapy at least for either of two reasons.
  • the variance may be associated with a specific disease subset that behaves differently with respect to one or more therapeutic interventions (i and ii above); second, the variance may affect response to a specific therapeutic intervention (iii above).
  • pharmacological therapeutic interventions In the first case, there may be no effect of a particular gene sequence variance on the observable pharmacological action of a drug, yet the disease subsets defined by the variance or variances differ in their response to the drug because, for example, the drug acts on a pathway that is more relevant to disease pathophysiology in one variance-defined patient subset thanin another variance-defined patient subset.
  • Useful gene sequence variances for this invention can be described as variances which partition patients into two or more groups that respond differently to a therapy, regardless of the reason for the difference, and regardless of whether the reason for the difference is known.
  • One exemplary method utilizes prior information on the pharmacology or pharmacokinetics or pharmacodynamics of a treatment method, e.g., the action of a drug, which indicates that a particular gene is, or is likely to be, involved in the action of the treatment method, and further suggests that variances in the gene may contribute to variable response to the treatment method.
  • variances in a gene can be co ⁇ elated empirically with treatment response.
  • variances in a gene which exist in a population can be identified.
  • the presence of the different variances or haplotypes in individuals of a study group, which is preferably representative of a population or populations, is determined.
  • This variance information is then co ⁇ elated with treatment response of the various individuals as an indication that genetic variability in the gene is at least partially responsible for differential treatment response.
  • Statistical measures known to those skilled in the art are preferably used to measure the fraction of inte ⁇ atient variation attributable to any one variance.
  • Useful methods for identifying genes relevant to the physiologic action of a drug or other treatment include large scale analysis of gene expression in cells treated with the drug compared to control cells, or large scale analysis of the protein expression pattern in treated vs. untreated cells, or the use of techniques for identification of interacting proteins or ligand-protein interactions.
  • the present invention generally concerns the identification of variances in genes which are indicative of the effectiveness of a treatment in a patient.
  • the identification of specific variances, in effect, can be used as a diagnostic or prognostic test.
  • Correlation of treatment efficacy and/or toxicity with particular genes and gene families or pathways is provided in Stanton et al., U.S. Provisional Application 60/093,484, filed July 20, 1998, entitled GENE SEQUENCE VARIANCES WITH UTILITY IN DETERMINING THE TREATMENT OF DISEASE (concerns the safety and efficacy of compounds active on folate or pyrimidine metabolism or action).
  • Genes identified in the examples below and the attached Tables and Figures can be used in the present invention.
  • the diagnostic test involves determining whether an individual has a variance or variant form of a gene that is involved in the disease or condition or the action of the drug or other treatment or effects of such treatment.
  • a variance or variant form of the gene is preferably one of several different variances or forms of the gene that have been identified within the population and are known to be present at a certain frequency.
  • the diagnostic test involves performed by amplifying a segment of DNA or RNA (generally after converting the RNA to cDNA) spanning one or more variances in the gene sequence.
  • the amplified segment is ⁇ 500 bases in length, in an alternative embodiment the amplified segment is ⁇ 100 bases in length, most preferably ⁇ 45 bases in length.
  • the diagnostic test is performed by amplifying a segment of DNA or RNA (cDNA) spanning a variance, or even spanning more than one variance in the gene sequence and preferably maintaining the phase of the variances on each allele.
  • phase means the association of variances on a single copy of the gene, such as the copy transmitted from the mother (maternal copy or maternal allele) or the father (paternal copy or paternal allele). It is apparent that such diagnostic tests are performed after initial identification of variances within the gene.
  • a genotyping test simply provides the status of a variance or variances in a subject or patient. For example suppose nucleotide 150 of hypothetical gene X on an autosomal chromosome is an adenine (A) or a guanine (G) base. The possible genotypes in any individual are AA, AG or GG at nucleotide 150 of gene X. In a haplotyping test there is at least one additional variance in gene X, say at nucleotide 810, which varies in the population as cytosine (C) or thymine (T).
  • C cytosine
  • T thymine
  • a particular copy of gene X may have any of the following combinations of nucleotides at positions 150 and 810: 150A-810C, 150A-810T, 150G-810C or 150G-810T.
  • Each of the four possibilities is a unique haplotype. If the two nucleotides interact in either RNA or protein, then knowing the haplotype can be important.
  • the point of a haplotyping test is to determine the haplotypes present in a DNA or cDNA sample (e.g. from a patient). In the example provided there are only four possible haplotypes, but, depending on the number of variances in the gene and their distribution in human populations there may be three, four, five, six or more haplotypes at a given gene.
  • the most useful haplotypes for this invention are those which occur commonly in the population being treated for a disease or condition. Preferably such haplotypes occur in at least 5% of the population, more preferably in at least 10%, still more preferably in at least 20% of the population and most preferably in at least 30% or more of the population. Conversely, when the goal of a pharmacogenetic program is to identify a relatively rare population that has an adverse reaction to a treatment, the most useful haplotypes may be rare haplotypes, which may occur in less than 5%, less than 2%, or even in less than 1% of the population.
  • the frequency of the adverse reaction will provide a useful guide to the likely frequency of salient causative haplotypes.
  • a diagnostic test utilizing methods known in the art can be used to determine whether a particular form of the gene, containing specific variances or haplotypes, or combinations of variances and haplotypes, is present in at least one copy, one copy, or more than one copy in an individual.
  • Such tests are commonly performed using DNA or RNA collected from blood, cells, tissue scrapings or other cellular materials, and can be performed by a variety of methods including, but not limited to, hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry or DNA sequencing, including minisequencing. Methods for haplotyping are provided in this application.
  • hybridization with allele specific probes can be conducted in two formats: (1) allele specific oligonucleotides bound to a solid phase (glass, silicon, nylon membranes) and the labelled sample in solution, as in many DNA chip applications, or (2) bound sample (often cloned DNA or PCR amplified DNA) and labelled oligonucleotides in solution (either allele specific or short so as to allow sequencing by hybridization).
  • a solid phase glass, silicon, nylon membranes
  • bound sample often cloned DNA or PCR amplified DNA
  • labelled oligonucleotides in solution either allele specific or short so as to allow sequencing by hybridization.
  • the present disclosure describes exemplary gene sequence variances in genes identified in a gene table herein (e.g., Tables 2, 6, and 8), and variant forms of these gene that may be determined using diagnostic tests.
  • a variance-based diagnostic test can be used to determine whether or not to administer a specific drug or other treatment to a patient for treatment of a disease or condition.
  • diagnostic tests are inco ⁇ orated in texts such as Clinical Diagnosis and Management by Laboratory Methods (19th Ed) by John B. Henry (Editor) W B Saunders Company, 1996; Clinical Laboratory Medicine : Clinical Application of Laboratory Data, (6th edition) by R.
  • Ravel, Mosby-Year Book, 1995, or medical textbooks including, without limitation, textbooks of medicine, laboratory medicine, therapeutics, pharmacy, pharmacology, nutrition, allopathic, homeopathic, and osteopathic medicine; most preferably such a diagnostic test is specified by regulatory authorities, e.g., by the U.S. Food and Drug Administration, and is inco ⁇ orated in the label or insert as well as the Physicians Desk Reference.
  • the procedure for using the drug is restricted or limited on the basis of a diagnostic test for determining the presence of a variance or variant form of a gene.
  • the procedure may include the route of administration of the drug, the dosage form, dosage, schedule of administration or use with other drugs; any or all of these may require selecting or determination consistent with the results of the diagnostic test or a plurality of such tests.
  • the use of such diagnostic tests to determine the procedure for administration of a drug is inco ⁇ orated in a text such as those listed above, or medical textbooks, for example, textbooks of medicine, laboratory medicine, therapeutics, pharmacy, pharmacology, nutrition, allopathic, homeopathic, and osteopathic medicine.
  • a diagnostic test or tests are required by regulatory authorities and are inco ⁇ orated in the label or insert as well as the Physicians Desk Reference.
  • Variances and variant forms of genes useful in conjunction with treatment methods may be associated with the origin or the pathogenesis of a disease or condition.
  • the variant form of the gene is associated with a specific characteristic of the disease or condition that is the target of a treatment, most preferably response to specific drugs or other treatments. Examples of diseases or conditions ameliorable by the methods of this invention are identified in the Examples and tables below; in general treatment of disease with cu ⁇ ent methods, particularly drug treatment, always involves some unknown element (involving efficacy or toxicity or both) that can be reduced by appropriate diagnostic methods.
  • the gene is involved in drug action, and the variant forms of the gene are associated with variability in the action of the drug.
  • one variant form of the gene is associated with the action of the drug such that the drug will be effective in an individual who inherits one or two copies of that form of the gene.
  • a variant form of the gene is associated with the action of the drug such that the drug will be toxic or otherwise contra-indicated in an individual who inherits one or two copies of that form of the gene.
  • diagnostic tests for variances and variant forms of genes as described above can be used in clinical trials to demonstrate the safety and efficacy of a drug in a specific population.
  • the drug is approved for sale or use by regulatory agencies with the recommendation or requirement that a diagnostic test be performed for a specific variance or variant form of a gene which identifies specific populations in which the drug will be safe and or effective.
  • the drug may be approved for sale or use by regulatory agencies with the specification that a diagnostic test be performed for a specific variance or variant form of a gene which identifies specific populations in which the drug will be toxic.
  • diagnostic tests for variances as described in this invention may be used in clinical trials to establish the safety and efficacy of a drug. Methods for such clinical trials are described below and/or are known in the art and are described in standard textbooks. For example, diagnostic tests for a specific variance or variant form of a gene may be inco ⁇ orated in the clinical trial protocol as inclusion or exclusion criteria for enrollment in the trial, to allocate certain patients to treatment or control groups within the clinical trial or to assign patients to different treatment cohorts.
  • diagnostic tests for specific variances may be performed on all patients within a clinical trial, and statistical analysis performed comparing and contrasting the efficacy or safety of a drug between individuals with different variances or variant forms of the gene or genes.
  • Prefe ⁇ ed embodiments involving clinical trials include the genetic stratification strategies, phases, statistical analyses, sizes, and other parameters as described herein.
  • diagnostic tests for variances can be performed on groups of patients known to have efficacious responses to the drug to identify differences in the frequency of variances between responders and non-responders.
  • diagnostic tests for variance are performed on groups of patients known to have toxic responses to the drug to identify differences in the frequency of the variance between those having adverse events and those not having adverse events.
  • outlier analyses may be particularly useful if a limited number of patient samples are available for analysis. It is apparent that such clinical trials can be or are performed after identifying specific variances or variant forms of the gene in the population.
  • the described approach and techniques are applicable to a variety of other diseases, conditions, and/or treatments and to genes associated with the etiology and pathogenesis of such other diseases and conditions and the efficacy and safety of such other treatments.
  • Useful variances for this invention can be described generally as variances which partition patients into two or more groups that respond differently to a therapy (a therapeutic intervention), regardless of the reason for the difference, and regardless of whether the reason for the difference is known.
  • a large set of exemplary variances are provided in Tables 3, 4, and 10; (3) using computational tools to predict the functional effects of variances in specific genes; (4) using in vitro or in vivo experiments to identify genes which may participate in the response to a drug or treatment, and to determine the variances which affect gene, RNA or protein function, and may therefore be important genetic variables affecting disease manifestations or drug response; and (5) retrospective or prospective clinical trials.
  • Tables 3, 4, and 10 A large set of exemplary variances are provided in Tables 3, 4, and 10; (3) using computational tools to predict the functional effects of variances in specific genes; (4) using in vitro or in vivo experiments to identify genes which may participate in the response to a drug or treatment, and to determine the variances which affect gene, RNA or protein function, and may therefore be important genetic variables affecting disease manifestations or drug response; and (5) retrospective or prospective clinical trials.
  • sources e.g., public databases and publications.
  • One skilled in the art can review the literature (textbooks, monographs, journal articles) and online sources (databases) to identify genes most relevant to the action of a specific drug or other treatment, particularly with respect to its utility for treating a specific disease, as this beneficially allows the set of genes to be analyzed ultimately in clinical trials to be reduced from an initial large set. Specific strategies for conducting such searches are described below.
  • the literature may provide adequate information to select genes to be studied in a clinical trial, but in other cases additional experimental investigations of the sort described below will be preferable to maximize the likelihood that the salient genes and variances are moved forward into clinical studies.
  • Experimental data are also useful in establishing a list of candidate genes, as described below.
  • the second step is to screen for variances in each candidate gene.
  • Experimental and computational methods for variance detection are described in this invention, and a tables of exemplary variances is provided (e.g., Table 3, 4, or 10) as well as methods for identifying additional variances.
  • the next step is to assess their likely contribution to clinical variation in patient response to therapy, preferably by using informatics-based approaches such as DNA and protein sequence analysis and protein modeling.
  • informatics-based approaches such as DNA and protein sequence analysis and protein modeling.
  • the literature and informatics-based approaches provide the basis for prioritization of candidate genes, however it may in some cases be desirable to further na ⁇ ow the list of candidate genes, or to measure experimentally the phenotype associated with specific variances or sets of variances (e.g. haplotypes).
  • (4) as a third step in candidate gene analysis, one skilled in the art may elect to perform in vitro or in vivo experiments to assess the functional importance of gene variances, using either biochemical or genetic tests.
  • the fourth step is to design retrospective or prospective human clinical trials to test whether the identified allelic variance, variances, or haplotypes or combination thereof influence the efficacy or toxicity profiles for a given drug or other therapeutic intervention. It should be recognized that this fourth step is the crucial step in producing the type of data that would justify introducing a diagnostic test for at least one variance into clinical use. Thus while each of the above four steps are useful in particular instances of the invention, this final step is indispensable. Further guidance and examples of how to perform these five steps is provided below.
  • Literature or online sources can provide specific genes involved in the disease process or drug response, or describe biochemical pathways involving multiple genes, each of which may affect the disease process or drug response.
  • biochemical or pathological changes characteristic of the disease may be described; such information can be used by one skilled in the art to infer a set of genes that can account for the biochemical or pathologic changes.
  • CNS central nervous system
  • genes responsible for serotonin biosynthesis release from the cell, receptor binding, presynaptic reuptake, and degradation or metabolism.
  • Genes responsible for each of these functions should be examined for variation that may account for inte ⁇ atient differences in drug response or disease manifestations.
  • Variation in a specific receptor may affect the pharmacology not only of drugs intentionally targeted to that receptor, but also drugs targeted to different receptors, that may have differential%ction on two allelic forms of the non-targeted receptor.
  • genes encoding proteins structurally related to the target protein are useful for screening for variance in the present invention.
  • a good general pharmacology text is Goodman & Gilman's the Pharmacological Basis of Therapeutics (9th Ed) by J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon and A.G. Gilman (Editors) McGraw Hill, 1996.
  • biomedical literature may include information on nonhuman organisms that is relevant to understanding the likely disease or pharmacological pathways in man.
  • DPD dihydropyrimidine dehydrogenase
  • the genetics of a rare allele or alleles may provide a basis for examining the effects of commonly occuring alleles on moderate phenotypes.
  • the genetics of rare DPD deficiency is well described in medical genetics textbooks listed below, for example see Scriver et al (full citation below).
  • OMIM Online Mendelian Inheritance in Man
  • the OMIM record number is provided for many of the genes in Table 10 (see column 3), and constitutes an excellent entry point for identification of references that point to the broader literature.
  • Another useful site at NCBI is the Entrez browser, located at http://www3.ncbi.nlm.nih.gov/Entrez .
  • More generally links to a number of useful sites with biomedical or genetic data are maintained at sites such as Med Web at the Emory University Health Sciences Center Library: http://WWW.MedWeb.Emorv.Edu/MedWeb/: Riken, a Japanese web site at: http://www.rtc.riken.go.ip/othersite.html with links to DNA sequence, structural, molecular biology, bioinformatics, and other databases; at the Oak Ridge National Laboratory web site: http://www.ornl.gov/hgmis/links.html: or at the Yahoo website of Diseases and Conditions: http://dir.yahoo.com/health/diseases and conditions/index.html. Each of the indicated web sites has additional useful links to other sites.
  • databases that provide information on biochemical pathways.
  • databases include the Kyoto Encyclopedia of Genes and Genomes (KEGG), which can be found at: http://www.genome.ad.ip kegg/kegg.html.
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • This site has pictures of many biochemical pathways, as well as links to other metabolic databases such as the well known Boehringer Mannheim biochemical pathways charts: http://www.expasv.ch cgi- bin/search-biochem-index.
  • the metabolic charts at the latter site are comprehensive, and excellent starting points for working out the salient enzymes on any given pathway.
  • Each of the web sites mentioned above has links to other useful web sites, which in turn can lead to additional sites with useful info ⁇ nation.
  • Biomedical Literature To obtain up to date information on drugs and their mechanism of action and biotransformation; disease pathophysiology; biochemical pathways relevant to drug action and disease pathophysiology; and genes that encode proteins relevant to drug action and disease one skilled in the art will consult the biomedical literature .
  • a widely used, publically accessible web site for searching published journal articles is PubMed (http://www.ncbi.nlm.nih.gov/PubMed/). At this site, one can search for the most recent articles (within the last 1-2 months) or for specific details on methods that are less recent (back to 1966). Many Journals also have their own sites on the world wide web and can be searched online.
  • RNA transcripts or proteins that are substantially increased or decreased in drug treated cells or tissues relative to control cells or tissues are candidates for mediating the action of the drug.
  • Other useful experimental methods include protein interaction methods such as the yeast two hybrid system and variants thereof which facilitate the detection of protein - protein interactions.
  • RNAs expressed in a cell is sometimes refe ⁇ ed to as the transcriptome.
  • Methods for measuring the transcriptome, or some part of it, are known in the art.
  • a recent collection of articles summarizing some cu ⁇ ent methods appeared as a supplement to the journal Nature Genetics. (The Chipping Forecast. Nature Genetics supplement, volume 21, January 1999.)
  • Experiments have been described in model systems that demonstrate the utility of measuring changes in the transcriptome before before and after changing the growth conditions of cells, for example by changing the nutritional status.
  • the changes in gene expression help reveal the network of genes that mediate physiological responses to the altered growth condition.
  • the addition of a drug to the cellular or in vivo environment, followed by monitoring the changes in gene expression can aid in identification of pharmacological gene networks.
  • the pool of proteins expressed in a cell is sometimes refe ⁇ ed to as the proteome.
  • Studies of the proteome may include not only protein abundance but also protein subcellular localization and protein-protein interaction.
  • Methods for measuring the proteome, or some part of it, are known in the art.
  • One widely used method is to extract total cellular protein and separate it in two dimensions, for example first by size and then by isoelectric point.
  • the resulting protein spots can be stained and quantitated, and individual spots can be excised and analyzed by mass spectrometry to provide definitive identification.
  • the results can be compared from two or more cell lines or tissues, at least one of which has been treated with a drug.
  • the differential up or down modulation of specific proteins in response to drug treatment may indicate their role in mediating the pharmaco logic actions of the drug.
  • Another way to identify the network of proteins that mediate the actions of a drug is to exploit methods for identifying interacting proteins.
  • a protein known to be involved in the action of a drug - for example the drug target - one can use systems such as the yeast two hybrid system and variants thereof (known to those skilled in the art) to identify additional proteins in the network of proteins that mediate drug action.
  • the genes encoding such proteins would be useful for screening for DNA sequence variances, which in turn may be useful for analysis of inte ⁇ atient variation in response to treatments.
  • the protein 5-lipoxygenase (5LO) s an enzyme which is a the beginning of the leukotriene biosynthetic pathway and is a target for anti-inflammatory drugs used to treat asthma and other diseases.
  • Genomic variance detection may include screening the entire genomic segment spanning the gene from the transcription start site to the polyadenylation site. Alternatively genomic variance detection may (for intron containing genes) include the exons and some region around them containing the splicing signals, for example, but not all of the intronic sequences.
  • promoter, enhancer, silencer and other regulatory elements have been described in human genes.
  • the promoter is generally proximal to the transcription start site, although there may be several promoters and several transcription start sites.
  • Enhancer, silencer and other regulatory elements may be intragenic or may lie outside the introns and exons, possibly at a considerable distance, such as 100 kb away.
  • Variances in such sequences may affect basal gene expression or regulation of gene expression. In either case such variation may affect the response of an individual patient to a therapeutic intervention, for example a drug, as described in the examples.
  • a prefe ⁇ ed method of variance detection is chain terminating DNA sequencing using dye labeled primers, cycle sequencing and software for assessing the quality of the DNA sequence as well as specialized software for calling heterozygotes.
  • the use of such procedures has been described by Nickerson and colleagues. See for example: Rieder M.J., et al. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res. 26 (4):967-73, 1998, and: Nickerson D.A., et al. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res.
  • variances e.g., provided in Tables 3, 4, and 10.
  • methods which can be used in the present invention for assessing the medical and pharmaceutical implications of a DNA sequence variance range from computational methods to in vitro and or in vivo experimental methods (discussed below), to prospective human clinical trials (see below), and also include a variety of other laboratory and clinical measures that can provide evidence of the medical consequences of a variance.
  • RNA or protein Phylogenetic approaches to understanding sequence variation are also useful. Thus if a sequence variance occurs at a nucleotide or encoded amino acid residue where there is usually little or no variation in homologs of the protein of interest from non- human species, particularly evolutionarily remote species, then the variance is more likely to affect function of the RNA or protein.
  • the appropriate biochemical assay may be to assess mRNA abundance, half life, or translational efficiency. If, on the other hand, there is no substantial evidence that the protein encoded by a particular gene is relevant to drug pharmacology, then the appropriate test is a clinical study addressing the responses to therapy of two patient groups distinguished on the basis of one or more variances. This approach reflects the cu ⁇ ent reality that biologists do not sufficiently understand gene regulation and gene expression to consistently make accurate inferences about the consequences of DNA sequence variances.
  • Variances in DNA may affect the basal transcription or regulated transcription of a gene locus. Such variances may be located in any part of the gene but are most likely to be located in the promoter region, the first intron, or in 5' or 3' flanking DNA, where enhancer or silencer elements may be located.
  • Methods for analyzing transcription are well known to those skilled in the art and exemplary methods are described in some of the texts cited below. Transcriptional run off assay is one useful method. Detailed protocols for useful methods can be found in texts such as: Cu ⁇ ent Protocols in Molecular Biology edited by: F.M. Ausubel, R.Brent, R.E. Scientific, D.D. Moore, J.G. Seidman, K.
  • RNA variances may affect a wide range of processes including RNA splicing, polyadenylation, capping, export from the nucleus, interaction with translation inflation, elongation or termination factors, or the ribosome, or interaction with cellular factors including regulatory proteins, or factors that may affect mRNA half life.
  • any effect of variances on RNA function should ultimately be measurable as an effect on RNA levels - either basal levels or regulated levels or levels in some abnormal cell state. Therefore one prefe ⁇ ed method for assessing the effect of RNA variances on RNA function is to measure the levels of RNA produced by different alleles in one or more conditions of cell or tissue growth.
  • Said measuring can be done by conventional methods such as Northern blots or RNAase protection assays (kits available from Ambion, Inc.), or by methods such as the Taqman assay (developed by the Applied Biosystems Division of the Perkin Elmer Co ⁇ oration), or by using a ⁇ ays of oligonucleotides or arrays of cDNAs attached to solid surfaces.
  • Systems for a ⁇ aying cDNAs are available commercially from companies such as Nanogen and General Scanning. Complete systems for gene expression analysis are available from companies such as Molecular Dynamics. For recent reviews of the technology see the supplement to volume 21 of Nature Genetics entitled "The Chipping Forecast" , especially articles begirining on pages 9, 15, 20 and 25.
  • RNA structural assays can be performed in vitro or on cell extracts or on
  • the prefe ⁇ ed method will depend on the availability of cells expressing a particular protein, and the feasibility of a cell-based assay vs. assays on cell extracts, on proteins produced in a foreign host, or on proteins prepared by in vitro translation.
  • the in vitro protein activity can be determined by transcription or translation in bacteria, yeast, baculovirus, COS cells (transient), CHO, or study directly in human cells. Further, one could perform pulse chase for experiments for the determination of changes in protein stability (half life).
  • One skilled in the art could manipulate the cell assay to address grouping the cells by genotypes or phenotypes. For example, identification of cells with different genotypes (possibly including families) and phenotype may be performed using standardized laboratory molecular biological protocols. After identification and grouping, one skilled in the art could determine whether there exists a co ⁇ elation between cellular genotype and cellular phenotype.
  • Advancing an experimental preclinical program may include testing these in vitro hypotheses in vivo, e.g. an animal model.
  • an embryonic stem cell is genetically manipulated to be deficient in a given gene. More specifically, a DNA construct is created that will undergo homologous recombination when inserted into the said embryonic stem cell nucleus. After the recombination event has occurred, the targeted gene is effectively inactivated due to the insertion of sequence (usually a translation stop or a marker gene sequence). This can be accomplished in worms, drosophila, or mice.
  • the species chosen will be conducive to attain maximal experimental results for the particular gene and the particular variance, variances, or haplotype. Once the knockout species is created the candidate therapeutic intervention can be administered to the animal and tested for effects on gene expression or effects of various gene deficiencies.
  • the chosen cell is a lower eukaryote, e.g. yeast
  • genetic manipulation occurs via introduction of a DNA construct that will undergo homologous recombination to disrupt the endogenous gene or genes.
  • a clinical trial is the definitive test of the utility of a variance or variances for the selection of optimal therapy.
  • Clinical trials require no knowledge of the biological function of the gene containing the variance or variances to be assessed, nor any knowledge of how the therapeutic intervention to be assessed works at a biochemical level; the question of the utility of a variance can be addressed at a purely phenomenological level.
  • a clinical trial can be designed to test a specific hypothesis.
  • a " clinical trial” is the testing of a therapeutic intervention in a volunteer human population for the pu ⁇ ose of determining whether a therapeutic intervention is safe and/or efficacious in the human volunteer or patient population for a given disease, disorder, or condition.
  • the analysis of safety and efficacy in genetically defined subgroups differing by at least one variance is of particular interest.
  • a " clinical study” is that part of a clinical trial that involves determination of the effect a candidate therapeutic intervention on human subjects. It includes clinical evaluations of physiologic responses including pharmacokinetic (abso ⁇ tion, distribution, bioavailability, and excretion) as well as pharmacodynamic (physiologic response and efficacy) parameters.
  • a pharmacogenetic clinical study is a clinical study that involves testing of one or more specific hypotheses regarding the effect of a genetic variance or variances (or set of variances, i.e. haplotype or haplotypes) in enrolled subjects or patients on response to a therapeutic intervention.
  • These hypotheses are articulated before the study in the form of primary or secondary endpoints.
  • the endpoint may be that in a particular genetic subgroup the rate of objectively defined responses exceeds some predefined threshold.
  • An Institutional Review Board accepts and reviews applications for clinical trials that are to be conducted at the institution and are to include healthy volunteers or human subjects from a defined patient population that seeks medical, surgical, rehabilitative, or social services at that institution.
  • the application includes document sections that provide the rationale for and describe the scope of the clinical study.
  • an application to an IRB may include a clinical protocol, and informed consent forms.
  • the supporting preclinical data is a report of all the in vitro, in vivo animal or previous human trial data that supports the safety and/or efficacy of a given therapeutic intervention.
  • the preclinical data may also include a description of the effect of a specific genetic variance or variances on biochemical or physiologic experimental variables, or on treatment outcomes, as determined by in vitro studies or by retrospective genetic analysis of clinical trial or other medical data (see below) used to first formulate or test a pharmacogenetic hypothesis.
  • the clinical protocol provides the relevant scientific and therapeutic introductory information, describes the inclusion and exclusion criteria for human subject enrollment, including genetic criteria if relevant, describes in detail the exact procedure or procedures for treatment using the candidate therapeutic intervention, describes laboratory analyses to be performed during the study period, and lastly describes the risks (both known and unknown) involving the use of the experimental candidate therapeutic intervention.
  • the clinical protocol will further describe the gene or genes believed or hypothesized to affect differential patient responses and the variance or variances to be tested.
  • the clinical protocol for a pharmacogenetic clinical trial will include a description of the stratification of the treatment groups based on one or more gene sequence variances or combination of variances or haplotypes.
  • the informed consent document is a description of the therapeutic intervention and the clinical protocol in simple language (third grade level) for the patient to read, understand, and, if willing, agree to participate in the study by signing the document.
  • the informed consent document will describe, in simple language, the use of a genetic test or a limited set of genetic tests to determine the subject or patients status at a particular gene variance or variances, and to further ascertain whether, in the study population, particular variances are associated with particular clinical or physiological responses.
  • the IND is composed of the investigator's brochure, the supporting in vitro and in vivo animal or previous human data, the clinical protocol, and the informed consent documents or forms.
  • a specific description of a single allelic variance or a number of variances to be tested in the clinical study will be included.
  • a description of the gene or genes believed or hypothesized to account, at least in part, for differential responses will be included as well as a description of genetic variance or variances of a particular candidate gene or genes.
  • the preclinical data may include a description of in vivo or in vitro studies of the biochemical or physiologic effects of a variance or variances (e.g., haplotype) in a candidate gene or genes, as well as the predicted effects of the variance or variances on efficacy or toxicology of the candidate therapeutic intervention.
  • a variance or variances e.g., haplotype
  • the results of retrospective genetic analysis of response data in patients treated with the candidate therapy may be the basis for formulating the genetic hypotheses to be tested in the prospective trial.
  • the focus of this section will be safety.
  • Phase I Phase I, II, III, and IV.
  • the fundamental objectives for each phase become increasingly complex as the stages of clinical development progress.
  • Phase I safety in humans is the primary focus.
  • dose-ranging designs establish whether the candidate therapeutic intervention is safe in the suspected therapeutic concentration range.
  • pharmacokinetic parameters e.g., adso ⁇ tion, distribution, metabolism, and excretion
  • pharmacokinetic parameters may be a secondary objective.
  • pharmacogenetic clinical study there may be additional analysis of the gene or genes and allelic variance or variances that are suspected to be involved in these pharmacokinetic parameters.
  • the dose or doses selected may be different than those identified based upon preclinical safety and efficacy determinations. For example, phenotypic effects of an allele depends on its frequency and also its interaction with the environment, as described earlier. Therefore, once the frequency of an allele or haplotype has been established for selected human subjects or patients, the effect of the variance on the drug responses by performing both in vitro or in vivo analyses under controlled conditions. Under these conditions, drug dosage could be adjusted accordingly.
  • the chosen dose may be one that is sub-optimal or is significantly less toxic so that determination of the effect of allelic variance or variances for a given treatment or human volunteer population may be appropriately tested and analyzed.
  • the dose may be similar to or the same as that chosen based upon in vitro or in vivo data.
  • the dose may be greater than optimal because allelic differences or haplotypes may result in enhanced elimination, metabolic inactivation, or excretion.
  • the numbers of individuals required for enrollment and the number of treatment conditions required to achieve the objectives of the trial is dictated by statistical power analysis.
  • the number of patients required for a given pharmacogenetic climcal trial will be determined on the prior knowledge of but not exclusively limited to variance or haplotype frequency, actual disease, disorder, or condition causing allele or allele associated with the disease, disorder, or condition and their linkage relationships.
  • the identified sample size will require an adequate analysis of the frequency of the allelic variance or variances within a given population, as described, for example, by Tu & Whitkemore (1999) and references therein.
  • Clinical trials can be designed to obscure the human subjects and/or the study coordinators from biasing that may occur during the testing of a candidate therapeutic invention.
  • the candidate therapeutic intervention is compared to best medical treatment, or a placebo (a compound, agent, device, or procedure that appears identical to the candidate therapeutic intervention but is innocuous to the receiving subject).
  • a placebo a compound, agent, device, or procedure that appears identical to the candidate therapeutic intervention but is innocuous to the receiving subject.
  • control with placebo limits efficacy perception by influencing factors such as prejudice on the part of the study participant or investigator, spontaneous alterations or variations that occur during treatment and are related to the disease studied, or are unrelated to the candidate therapeutic intervention.
  • a placebo arm or best medical therapy may be required in order to ascertain the effect of the allelic variance or variances on the efficacy or toxicology of the candidate therapeutic intervention.
  • Blinding refers to the lack of knowledge of the identity of the trial treatment and thus can be used to ascertain the real and not perceived effects of the candidate therapeutic intervention.
  • Patients, trial subjects, investigators, data review committees, ancillary personnel, statisticians, and clinical trial monitors may be blinded or unblinded during the trial period.
  • Open label trials refer to those that are unblinded; single blind is when the patient is kept unaware of the treatment groups; double blind is when both the patient and the investigator is kept unaware of the treatment groups; or a combination of these may be instituted during the trial period.
  • Pharmacogenetic clinical trial design may include one or a combination of open label, single blind, or double blind clinical trial design because reduction of inherent biases due to the knowledge of the type of treatment the human subject or the patient is to receive will ensure detection of the accuracy of the benefits of the stratification based upon allelic variance or variances or haplotypes.
  • termination endpoints for trials including or excluding pharmacogenetic objectives are defined and include observation of adverse clinical events, voluntary lack of study participation either in the form of lack of adherence to the clinical protocol or sudden change in lifestyle of the participant, lack of adherence on the part of trial investigators to follow the trial protocol, death, or lack of efficacy or positive response within the test group.
  • Phase I of clinical development is a safety study performed in a limited ( ⁇ 15) number of normal, healthy volunteers usually at single institutions. The primary endpoints in these studies is to determine pharmacokinetic parameters (i.e. adso ⁇ tion, distribution, and bioavailability), dose-related side effects that are either desirable or undesirable, and metabolites that co ⁇ oborate preclinical animal studies.
  • stratification based upon allelic variance or variances of a suspected gene or genes involving any or all of the pharmacokinetic parameters will be considered and inco ⁇ orated in the objectives of the trial design.
  • a pharmacogenetic Phase I study may enroll healthy human volunteers and stratify these individuals based upon their genotype.
  • a study objective may include observation of the effect of the allele/haplotype (detectable or undetectable) which the candidate therapeutic intervention may exhibit within the allelic variance, allelic variances, or haplotype groupings which can be assessed in the absence of a disease, disorder, or condition.
  • Phase I studies can include a limited number of patients with a diagnosed disease, disorder, or condition for whom clinical parameters satisfy a specified inclusion criteria (see below). These safety/limited efficacy studies can be conducted at multiple institutions to ensure enrollment of these patients. In a pharmacogenetic Phase I study that will include patients to some degree, the gene or genes and allelic variance or variances suspected to be involved in the efficacy of the candidate therapeutic intervention will be considered in the design of the inclusion criteria, the objectives, and the primary endpoints.
  • Phase II studies include a limited number of patients ( ⁇ 100) that satisfy the required inclusion criteria and do not satisfy any of the exclusion criteria of the trial design. Phase II studies can be conducted at single or multiple institutions.
  • Inclusion criteria for patient enrollment to a clinical trial is a list of qualities for a given patient population that includes pathophysiologic clinical parameters for a given disease, disorder, or condition that can be determined by clinical diagnosis or laboratory or diagnostic test; age; gender; fertility state (e.g. pre- or postmenopausal women); coexisting medical therapies; or psychological, emotional, or cognitive state.
  • Inclusion criteria can also include defined psychological, emotional, or socioeconomic support by family or friends.
  • Exclusion criteria for patient enrollment generally includes the listing of co-morbidities that may interfere with the observations of the medical or laboratory pathophysiological clinical parameters of the disease, disorder, or condition, age, gender, fertility state (e.g. pre- or postmenopausal women), or previous or concu ⁇ ent medical, surgical, or diagnostic therapies.
  • the primary endpoint of the study is generally limited efficacy and co ⁇ oboration of the Phase I safety data in the specified patient population defined by the inclusion/exclusion criteria of the clinical protocol.
  • Primary efficacy endpoints include observed improvements of pathophysiologic parameters that are determined medically, diagnostically (e.g. clinical laboratory values), or by surrogate measurements of the pathological state of the disease, disorder, or condition.
  • Primary endpoints may also include limitation of pharmacologic therapies, reduction of time to death, or reduction in the progression of the disease, disorder, or condition.
  • Surrogate markers are pathophysiologic parameters determined by medical or climcal laboratory diagnosis that are associated and have been co ⁇ elated with the prognosis, progression, predisposition, or risk analysis with a disease, disorder, or condition that are not directly related to the primary diagnosed pathophysiologic condition, e.g. lowering blood pressure and coronary heart disease.
  • Secondary endpoints are those that supplement the primary endpoint and can be used to support further clinical studies. For example, secondary endpoints include reduction in pharmacologic therapy, reduction in requirement of a medical device, or alteration of the progression of the disease disorder, or condition.
  • Treatment groups with varying doses are included in the study to identify the appropriate dosage and pharmacokinetic parameters to achieve maximum efficacy.
  • pharmacodynamic parameters may include surrogate endpoints, efficacy endpoints, or pathophysiologic thresholds.
  • Pharmacokinetic parameters may include but are not exclusive of dosage, toxicological variables, metabolism, or excretion. Other parameters that may effect the outcome of a pharmacogenetic clinical trial may include gender, race, ethnic origins (population history), and combination of allelic variances of genes from multiple pathways, leading to but not exclusively efficacy or toxicology.
  • Phase III studies include multi-site, large, statistically significant, numbers of patients ( ⁇ 5,000) that fulfill the inclusion criteria for the study.
  • the design of this type of trial includes power analysis to ensure the data will support the study objectives.
  • the primary endpoint is preferably defined as enhanced efficacy as compared to placebo or best medical care for said disease, disorder, or condition.
  • the primary endpoint may include reduction of condition progression, improvement of a specific subset of symptoms, or in requirement or perceived need of medical therapy.
  • the endpoints will be the determination of the efficacy or toxicological differences that can be demonstrated to be dependent on the stratification based upon allelic variance or variances in a gene or genes that are suspected to be involved in the efficacy or toxicological population phenotype. Further in the Phase III pharmacogenetic clinical trial, the analysis of the impact of the allelic variance or variances will be broadened from the confirmatory Phase II pharmacogenetic clinical trial data that supports the notion that the phenotypic response differences can be identified as dependent on the allelic variance or variances of a gene or genes suspected to be involved in the efficacy or toxicological response.
  • the NDA includes the raw (unanalyzed) clinical data, i.e. the primary endpoints or secondary endpoints, a statistical analysis of all of the included data, a document describing in detail any adverse or observed side effects, tabulation of the participant drop-outs and detailed reasons for the termination, and other specific data or details of ongoing in vitro or in vivo studies since the submission of the IND. If pharmacoeconomic objectives are a part of the clinical trial design data supporting cost or economic analyses are included in the NDA.
  • the pharmacoeconomic analyses may include demonstration or lack of benefit of the candidate therapeutic intervention in a cost benefit analysis, cost of illness study, cost minimization study, or cost utility analysis.
  • the effect of a diagnostic identification of the population and subsequent stratification based upon allelic variance or variances or haplotype of a suspected gene or genes involved in the efficacy or toxicological responses of the candidate therapeutic intervention will be used to support application for the approval for the marketing and sale of the candidate therapeutic intervention.
  • Phase IV studies occur after the therapeutic intervention has been approved for marketing. In these studies, retrospective data and data from a large patient population that do not necessarily fulfill the pathophysiologic requirements of the approved indication are included.
  • both retrospective and prospective design can be inco ⁇ orated. In both cases, stratification based upon allelic variance or variances with adequate sample size in order to determine the statistical relevance of an outcome difference among the treatment groups.
  • Phase I, II, III development i.e. the clinical development of candidate therapeutic interventions for serious debilitating or life threatening diseases, or for those cases whereby no medical therapeutic alternative exists.
  • target indication for cancer or medically intractable, life threatening or seriously debilitating diseases, disorders, or conditions the US FDA has regulatory procedural mechanisms that can expedite the availability of the therapeutic intervention for patients that fall into one or more of these categories.
  • development incentives include Treatment IND, Fast-Track or Accelerated review, and O ⁇ han Drug Status.
  • allelic variance or variances may have on the outcome response or endpoints is inco ⁇ orated. Further consideration may include but is not limited to accrual rate for candidate patients, and number of institutions or climcal sites required to achieve an appropriate sample size.
  • sponsors may choose to expedite the development of the candidate therapeutic intervention without making use of the above FDA regulatory clinical development incentives.
  • the sponsor proposes expedited clinical development of a candidate therapeutic intervention due to outstanding positive or unequivocal preclinical safety and/or efficacy data.
  • supplemental applications are those in which a candidate therapeutic intervention is tested in a human clinical trial in order for the product to have an expanded label to include additional indications for therapeutic use.
  • the previous clinical studies of the therapeutic intervention i.e. those involving the preclinical safety and Phase I human safety studies can be used to support the testing of the particular candidate therapeutic intervention in a patient population for a different disease, disorder, or condition than that previously approved in the US.
  • a limited Phase II study is performed in the proposed patient population. With adequate signs of efficacy, a Phase III study is designed. All other parameters of clinical development for this category of candidate therapeutic interventions proceeds as described above for interventions first tested in human candidates.
  • outcomes or “therapeutic outcomes” are used to describe the results and value of healthcare intervention. Outcomes can be multi-dimensional, e.g., including one or more of the following: improvement of symptoms; regression of the disease, disorder, or condition; economic outcomes of healthcare decisions.
  • pharmacoeconomics is the analysis of a therapeutic intervention in a population of patients diagnosed with a disease, disorder, or condition that includes at least one of the following studies: cost of illness study (COI); cost benefit analysis (CBA), cost minimization analysis (CMA), or cost utility analysis (CUA), or an analysis comparing the relative costs of a therapeutic intervention with one or a group of other therapeutic interventions.
  • costs are those economic variables associated with a disease, disorder, or condition fall into two broad categories: direct and indirect.
  • Direct costs are associated with the medical and non-medical resources used as therapeutic interventions, including medical, surgical, diagnostic, pharmacologic, devices, rehabilitation, home care, nursing home care, institutional care, and prosthesis.
  • Indirect costs are associated with loss of productivity due to the disease, disorder, or condition suffered by the patient or relatives.
  • health-related quality of life is a measure of the impact of the disease, disorder, or condition on an individual's or group of patient's activities of daily living.
  • included in pharmacoeconomic studies is an analysis of the health- related quality of life. Standardized surveys or questionnaires for general health-related quality of life or disease, disorder, or condition specific determine the impact the disease, disorder, or condition has on an individuals day to day life activities or specific activities that are affected by a particular disease, disorder, or condition.
  • stratification refers to the creation of a distinction between patients on the basis of a characteristic or characteristics of the patient. Generally, in the context of clinical trials, the distinction is used to distinguish responses or effects in different sets of patients distinguished according to the stratification parameters.
  • stratification preferably includes distinction of patient groups based on the presence or absence of particular variance or variances in one or more genes. The stratification may be performed only in the course of analysis or may be used in creation of distinct groups or in other ways.
  • a human clinical trial can result in data to support the utility of a gene variance or variances for the selection of optimal therapy.
  • Climcal studies require no knowledge of the biological function of the gene containing the variance of the variances to be assessed, nor any knowledge of how the therapeutic invention to be assessed works at a biochemical level.
  • the data sets include one or a combination of at least of the following:
  • the target genes can be appropriately identified.
  • the candidate therapy e.g. drug
  • the target genes can be appropriately identified.
  • In vitro data supporting altered physiologic activity of the variant forms of the gene in the presence of the therapy assists the direction of the fundamental hypotheses and identifying the objectives for a human clinical trial.
  • the combined preclinical data sets should point to the premise of a controlled clinical trial of the the therapeutic intervention.
  • the design of the trial will preferably inco ⁇ orate the preclinical data sets to determine the primary and secondary endpoints.
  • these endpoints will include whether the therapeutic intervention is efficacious, efficacious with undesirable side effects, ineffective, ineffective with undesirable side effects, or ineffective with deleterious effects.
  • Pharmacoeconomic analyses may be inco ⁇ orated in order to support the efficacious intervention, efficacious with undesirable side effects cases, whereby the clinical outcome is positive, and economic analyses are required for the support of overall benefit to the patient and to society.
  • the strategies for designing a clinical trial to test the effect of a genotypic variance or variances on a physiological response to therapeutic intervention for drugs with known mechanism of action, mechanism of biotransformation, and/or known physiologic response differentials co ⁇ elated to genotypic variance or variances will be modified based upon the data and information from the preclinical studies and the patient symptomatic parameters unique to the target indication.
  • the strategy (design) and the implementation (conduct) of the clinical study preferably consist of one or more of the following strategies.
  • retrospective clinical trials will be to test and refine hypotheses regarding genetic factors that are associated with drug responses.
  • the best supported hypotheses can subsequently be tested in prospective clinical trials, and data from the prospective trials will likely comprise the main basis for an application to register the drug and predictive genetic test with the appropriate regulatory body. In some cases, however, it may become acceptable to use data from retrospective trials to support regulatory filings.
  • Genetic stratification of patients can be accomplished in several ways, including the following (where 'A' is the more frequent form of the variance being assessed and 'a' is the less frequent form):
  • the effect of genotype on drug response phenotype may be affected by a variety of nongenetic factors. Therefore it may be beneficial to measure the effect of genetic stratification in a subgroup of the overall clinical trial population.
  • Subgroups can be defined in a number of ways including, for example, biological, climcal, pathological or environmental criteria.
  • the predictive value of genetic stratification can be assessed in a subgroup or subgroups defined by: a.
  • Biological criteria i. gender (males vs. females) ii. age (for example above 60 years of age). Two, three or more age groups may be useful for defining subgroups for the genetic analysis. iii.
  • the status of Alzheimer's Disease patients is often measured by cognitive assessment scales such as the mini-mental status exam (MMSE) or the Alzheimer's Disease Assessment Scale (ADAS), which includes a cognitive component (ADAS-COG).
  • MMSE mini-mental status exam
  • ADAS Alzheimer's Disease Assessment Scale
  • ADAS-COG cognitive component
  • pathological criteria i. Histopathologic features of disease tissue, or pathological diagnosis.
  • lung cancer squamous cell carcinoma, adenocarcinoma, small cell carcinoma, bronchoalveolar carcinoma, etc., each of which may - which, in combination with genetic variation, may co ⁇ elate with ii. Pathological stage.
  • a variety of diseases have pathological staging schemes iii. Loss of heterozygosity (LOH) iv.
  • Pathology studies such as measuring levels of a marker protein v. Laboratory studies such as hormone levels, protein levels, small molecule levels
  • Subgroups may be defined in several ways, i. more than two age groups ii. age related status such as pre or post-menopausal Stratify by haplotype at one candidate locus where the haplotype is made up of two variances, three variances or greater than three variances.
  • Tested distributions can include extreme value, normal and logistic distributions, and, by using a log transformation, exponential, Weibull, lognormal, loglogistic and gamma distributions.
  • Identify variances in the candidate genes Initially, individual variances (and preferably their frequencies) will be identified by standard methods. Then, for genes with more than one variance, the commonly occurring patterns of variances occurring on a single chromosome (i.e. the haplotypes) may also be established using both computational and experimental approaches. For example, a computational approach might include one of, but not limited to, the following two methods a) expectation maximization (E-M) algorithm (Excoffier and Slatkin, Mol. Biol. Evol. 1995) and, b) a combination of Parsimonious and E-M methods. If we have a large population, implementation of the E-M method will be performed first.
  • E-M expectation maximization
  • a given phenotype or a sequence could come from several genotypes. This is particularly true if the sequence is heterozygous at a number of nucleotide 5 positions. Therefore, it is not practical to just count the phenotypes and make a conclusion on the underlying genotype, because it may lead to ambiguities. To avoid such ambiguities, an alternative iterative method called the EM (expectation- maximization) algorithm is used to derive the expected genotypes for a given phenotype or a sequence. This method assumes that the population under 10 consideration is in Hardy- Weinberg equilibrium.
  • the expected number of genotypic frequencies in the population is in H-W equilibrium for any given (all) allele(s) frequency. This is followed by setting the allele frequencies and iteration n, and testing for its stability in a series of iterations, up to m. When the values of the 20 initial allele frequencies stabilize at the end of series of iterations up to m, the resulting expected number of genotypes are assigned to phenotypes; for example, sequences or individuals.
  • parsimony methods As indicated above, also among the number of methods which are used for the pu ⁇ ose of classifying D ⁇ A sequences, haplotypes or phenotypic characters are the parsimony methods. Parsimony principle maintains that the best explanation for the observed differences among sequences, phenotypes (individuals, species) etc., is provided by the smallest number of evolutionary changes. Alternatively, simpler hypotheses are preferable to explain a set of data or patterns, than more complicated ones, and that ad hoc hypotheses should be avoided whenever possible (Molecular Systematics, Hillis et al., 1996). These methods for inferring relationship among sequences operate by minimizing the number of evolutionary steps or mutations (changes from one sequence/character) required to explain a given set of data.
  • supposing we want to obtain relationships among a set of sequences and construct a structure (tree/topology), we first count the minimum number of mutations that are required for explaining the observed evolutionary changes among a set of sequences. A structure (topology) is constructed based on this number. When once this number is obtained, another structure is tried. This process is continued for all reasonable number of structures. Finally, the structure that required the smallest number of mutational steps is chosen as the likely structure/evolutionary tree for the sequences studied.
  • the computed frequency of the haplotypes are equal to the number of individuals in the population, then there will be a consideration of utilizing additional methods. For these cases and if there is a small population, then the number of haplotypes will be considered relative to the number of entrants. In a method that is a modification of previously published work (Clark, Mol Biol and Evol. 1990) homozygotes will be assigned one unambiguous haplotype. If there is a single site variance (mutation) at one of the chromosomes then it will have two haplotypes. As the number of variances (mutations) increase in the diploid chromosomes, each of these variances will be compared with the haplotypes of the original population. Then a frequency will be assigned to the new variance based upon the Hardy- Weinberg expected frequencies. (See text below for why haplotypes are useful and how to determine them experimentally, if necessary.)
  • phase III trials with selected variances as inclusion criteria and clinical/pharmacoeconomic endpoints.
  • the number of patients required for adequate statistical power (approximately the same as in a usual phase III trial) will be determined from the phase II results and allele frequencies.
  • phase III trials with selected variances as inclusion criteria and clinical/pharmacoeconomic endpoints.
  • the number of patients required for adequate statistical power (approximately the same as in a usual phase III trial) will be determined from the phase II results and allele frequencies.
  • a clinical trial in which pharmacogenetic related efficacy or toxicity endpoints are included in the primary or secondary endpoints will be part of a retrospective or prospective clinical trial.
  • allelic differences will be identified and stratification based upon these genotypic differences among patient or subject groups will be used to ascertain the significance of the impact a genotype has on the candidate therapeutic intervention.
  • Retrospective pharmacogenetic trials can be conducted at each of the phases of clinical development, with the assumption that sufficient data is available for the co ⁇ elation of the physiologic effect of the candidate therapeutic intervention and the allelic variance or variances within the treatment population.
  • the data collected from the trial can be re-analyzed by imposing the additional stratification on groups of patients by specific allelic variances that may exist in the treatment groups.
  • Retrospective trials can be useful to ascertain whether a hypothesis that a specific variance has a significant effect on the efficacy or toxicity profile for a candidate therapeutic intervention.
  • a prospective clinical trial has the advantage that the trial can be designed to ensure the trial objectives can be met with statistical certainty.
  • power analysis which includes the parameters of allelic variance frequency, number of treatment groups, and ability to detect positive outcomes can ensure that the trial objectives are met.
  • Phase III clinical data can indicate trial variables for which further analysis is required. For example, su ⁇ ogate endpoints, pharmacokinetic parameters, dosage, efficacy endpoints, ethnic and gender differences, and toxicological parameters may result in data that would require further analysis and re-examination through the design of an additional trial. In these cases, analysis involving statistics,, genetics, clinical outcomes, and economic parameters may be considered prior to proceeding to the stage of designing any additional trials. Factors involved in the consideration of statistical significance may include Bonfe ⁇ oni analysis, pe ⁇ nutation testing, with multiple testing co ⁇ ection resulting in a difference among the treatment groups that has occurred as a result of a chance of no greater than 20%, i.e. p ⁇ 0.20.
  • Factors included in determining clinical outcomes to be relevant for additional testing may include, for example, consideration of the target indication, the trial endpoints, progression of the disease, disorder, or condition during the trial study period, biochemical or pathophysiologic relevance of the candidate therapeutic intervention, and other variables that were not included or anticipated in the initial study design or clinical protocol.
  • Factors to be included in the economic significance in determining additional testing parameters include sample size, accrual rate, number of clinical sites or institutions required, additional or other available medical or therapeutic interventions approved for human use, and additional or other available medical or therapeutic interventions concu ⁇ ently or anticipated to enter human clinical testing.
  • a prospective clinical trial having an objective to determine the significance of the variable or parameter and its effect on the outcome of the parent Phase II trial.
  • a pharmacogenetic difference i.e. a single or multiple allelic difference
  • a population could be selected based upon the distribution of genotypes.
  • the candidate therapeutic intervention could then be tested in this group of volunteers to test for efficacy or toxicity.
  • the repeat prospective study could be a Phase I limited study in which the subjects would be healthy human volunteers, or a Phase II limited efficacy study in which patients which satisfy the inclusion criteria could be enrolled. In either case, the second, confirmatory trial could then be used to systematically ensure an adequate number of patients with appropriate phenotype is enrolled in a Phase III trial.
  • a placebo controlled pharmacogenetics clinical trial design will be one in which target allelic variance or variances will be identified and a diagnostic test will be performed to stratify the patients based upon presence, absence, or combination thereof of these variances.
  • determination of a specific sample size of a prospective trial will be described to include factors such as expected differences between a placebo and treatment on the primary or secondary endpoints and a consideration of the allelic frequencies.
  • the design of a pharmacogenetics clinical trial will include a description of the allelic variance impact on the observed efficacy between the treatment groups.
  • the type of genetic and phenotypic relationship display of the efficacy response to a candidate therapeutic intervention will be analyzed.
  • a genotypically dominant allelic variance or variances will be those in which both heterozygotes and homozygotes will demonstrate a specific phenotypic efficacy response different from the homozygous recessive genotypic group.
  • a pharmacogenetic approach is useful for clinicians and public health professionals to include or eliminate small groups of responders or non-responders from treatment in order to avoid unjustified side-effects. Further, adjustment of dosages when clear clinical difference between heterozygous and homozygous individuals may be beneficial for therapy with the candidate therapeutic intervention
  • a reccesive allelic variance or variances will be those in which only the homozygote recessive for that or those variances will demonstrate a specific phenotypic efficacy response different from the heterozygotes or homozygous dominants.
  • An extension of these examples may include allelic variance or variances organized by haplotypes from additional gene or genes providing an explanation of clinical phenotypic outcome differences among the treatment groups. These types of clinical studies will point and address allelic variance and its role in the efficacy or toxicology pattern within the treatment population.
  • the table above shows the probability (expressed as percent) of detecting both alleles (i.e. detecting heterozygotes) at a biallelic locus as a function of (i) the allele frequencies and (ii) the number of individuals genotyped.
  • the chances of detecting heterozygotes increases as the frequencies of the two alleles approach 0.5 (down a column), and as the number of individuals genotyped increases (to the right along a row).
  • the numbers in the table are given by the formula: 1 - (p) Allele frequencies are designated p and q and the number of individuals tested is designated n.
  • Nucleic acid samples for example for use in variance identification, can be obtained from a variety of sources as known to those skilled in the art, or can be obtained from genomic or cDNA sources by known methods.
  • the Coriell Cell Repository (Camden, N.J.) maintains over 6,000 human cell cultures, mostly fibroblast and lymphoblast cell lines comprising the NIGMS Human Genetic Mutant Cell Repository.
  • a catalog http://locus.umdnj.edu/nigms) provides racial or ethnic identifiers for many of the cell lines. 55 of the 62 cell lines to be genotyped (as indicated above) are drawn from this collection; the remainder were obtained from the Beijing Cancer Institute.
  • the cell lines are derived from 21 Caucasians (of Northern, Central and Southern European origin), 8 Afro-Americans, 9 Hispanics or Mexicans, 8 Chinese, 12 Japanese, 1 American Indian, 1 East Indian, 1
  • Source of human DNA, RNA and cDNA samples PCR based screening for DNA polymo ⁇ hism can be carried out using either genomic DNA or cDNA produced from mRNA.
  • genomic DNA has the advantage that variances can be identified in promoter, intron and flanking regions. Such variances may be biologically relevant. Therefore preferably, when variance analysis of patients with outlier responses is performed, analysis of selected loci at the genomic level is also performed. Such analysis would be contingent on the availability of a genomic sequence or intron-exon boundary sequences, and would also depend on the anticipated biological importance of the gene in connection with the particular response.
  • cDNA When cDNA is to be analyzed it is very beneficial to establish a tissue source in which the genes of interest are expressed at sufficient levels that cDNA can be readily produced by RT-PCR. Preliminary PCR optimization efforts for 19 of the 29 genes in Table 2 reveal that all 19 can be amplified from lymphoblastoid cell mRNA. The 7 untested genes belong on the same pathways and are expected to also be PCR amplifiable.
  • Primers for amplifying a particular sequence can be designed by methods known to those skilled in the art, including by the use of computer programs such as the PRIMER software available from Whitehead
  • T4 EndoVII The enzyme T4 endonuclease VII (T4E7) is derived from the bacteriophage T4. T4E7 specifically cleaves heteroduplex DNA containing single base mismatches, deletions or insertions. The site of cleavage is 1 to 6 nucleotides 3' of the mismatch. This activity has been exploited to develop a general method for detecting DNA sequence variances (Youil et al. 1995; Mashal and Sklar, 1995).
  • T4E7 variance detection procedure based on the T4E7 patent of R.G.H. Cotton and co-workers. (Del Tito et al., in press) is preferably utilized.
  • T4E7 has the advantages of being rapid, inexpensive, sensitive and selective. Further, since the enzyme pinpoints the site of sequence variation, sequencing effort can be confined to a 25 -30 nucleotide segment.
  • the major steps in identifying sequence variations in candidate genes using T4E7 are: (1) PCR amplify 400-600 bp segments from a panel of DNA samples; (2) mix a fluorescently-labeled probe DNA with the sample DNA; (3) heat and cool the samples to allow the formation of heteroduplexes; (4) add T4E7 enzyme to the samples and incubate for 30 minutes at 37°C, during which cleavage occurs at sequence variance mismatches; (5) run the samples on an ABI 377 sequencing apparatus to identify cleavage bands, which indicate the presence and location of variances in the sequence; (6) a subset of PCR fragments showing cleavage are sequenced to identify the exact location and identity of each variance.
  • the T4E7 Variance Imaging procedure has been used to screen particular genes.
  • the efficiency of the T4E7 enzyme to recognize and cleave at all mismatches has been tested and reported in the literature.
  • One group reported detection of 81 of 81 known mutations Youil et al. 1995
  • another group reported detection of 16 of 17 known mutations Merashal and Sklar, 1995.
  • the T4E7 method provides highly efficient variance detection.
  • a subset of the samples containing each unique T4E7 cleavage site is selected for sequencing.
  • DNA sequencing can , for example, be performed on ABI 377 automated DNA sequencers using BigDye chemistry and cycle sequencing. Analysis of the sequencing runs will be limited to the 30-40 bases pinpointed by the T4E7 procedure as containing the variance. This provides the rapid identification of the altered base or bases.
  • the presence of variances can be infe ⁇ ed from published articles which describe Restriction Fragment Length Polymo ⁇ hisms (RFLP).
  • the sequence variances or polymo ⁇ hisms creating those RFLPs can be readily determined using convention techniques, for example in the following manner.
  • the molecular sequence of the RFLP can be determined by restricting the cDNA probe into fragments and separately hybridizing to a Southern blot consisting of the restriction digestion with the enzyme which reveals the polymo ⁇ hic site, identifying the sub-fragment which hybridizes to the polymo ⁇ hic restriction fragment, obtaining a genomic clone of the gene (e.g., from commercial services such as Genome Systems (Saint Louis, Missouri) or Research Genetics (Alabama) which will provide appropriate genomic clones on receipt of appropriate primer pairs).
  • a genomic clone of the gene e.g., from commercial services such as Genome Systems (Saint Louis, Missouri) or Research Genetics (Alabama) which will provide appropriate genomic clones on receipt of appropriate primer pairs.
  • variances can be detected using computational methods, involving computer comparison of sequences from two or more different biological sources, which can be obtained in various ways, for example from public sequence databases.
  • the term "variance scanning” refers to a process of identifying sequence variances using computer-based comparison and analysis of multiple representations of at least a portion of one or more genes.
  • Computational variance detection involves a process to distinguish true variances from sequencing e ⁇ ors or other artifacts, and thus does not require perfectly accurate sequences. Such scanning can be performed in a variety of ways as known to those skilled in the art, preferably, for example, as described in Stanton and Adams, U.S. Patent Application filed April 26, 1999, serial number not yet assigned, attorney docket 241/034.
  • RNA sequences may be provided as RNA sequences, e.g., mRNA sequences. Such RNA sequences may be converted to the co ⁇ esponding DNA sequences, or the analysis may use the RNA sequences directly.
  • probes include nucleic acid hybridization probes and antibodies, for example, monoclonal antibodies, which can differentially bind to nucleic acid sequences differing in one or more variance sites or to polypeptides which differ in one or more amino acid residues as a result of the nucleic acid sequence variance or variances. Generation and use of such probes is well-known in the art and so is not described in detail herein.
  • the presence or absence of a variance is determined using nucleotide sequencing of a short sequence spanning a previously identified variance site.
  • This will utilize validated genotyping assays for the polymo ⁇ hisms previously identified. Since both normal and tumor cell genotypes can be measured, and since tumor material will frequently only be available as paraffin embedded sections (from which RNA cannot be isolated), it will be necessary to utilize genotyping assays that will work on genomic DNA.
  • PCR reactions will be designed, optimized, and validated to accommodate the intron exon structure of each of the genes. If the gene structure has been published (as it has for some of the listed genes), PCR primers can be designed directly.
  • PCR primers may need to be moved around in order to both span the variance and avoid exon-intron boundaries.
  • one-sided PCR methods such as bubble PCR (Ausubel et al. 1997) may be useful to obtain flanking intronic DNA for sequence analysis.
  • the standard method used to genotype normal and tumor tissues will be DNA sequencing. PCR fragments encompassing the variances will be cycle sequenced on ABI 377 automated sequencers using Big Dye chemistry
  • a differential treatment response Prior to establishment of a diagnostic test for use in the selection of a treatment method or elimination of a treatment method, the presence or absence of one or more specific variances in a gene or in multiple genes is co ⁇ elated with a differential treatment response. (As discussed above, usually the existence of a variable response and the co ⁇ elation of such a response to a particular gene is performed first.) Such a differential response can be determined using prospective and/or retrospective data. Thus, in some cases, published reports will indicate that the course of treatment will vary depending on the presence or absence of particular variances. That information can be utilized to create a diagnostic test and/or inco ⁇ orated in a treatment method as an efficacy or safety determination step. Usually, however, the effect of one or more variances is separately determined.
  • the determination can be performed by analyzing the presence or absence of particular variances in patients who have previously been treated with a particular treatment method, and co ⁇ elating the variance presence or absence with the observed course, outcome, and/or development of adverse events in those patients. This approach is useful in cases where both the observation of treatment effects was clearly recorded and cell samples are available or can be obtained. Alternatively, the analysis can be performed prospectively, where the presence or absence of the variance or variances in an individual is determined and the course, outcome, and/or development of adverse events in those patients is subsequently or concurrently observed and then co ⁇ elated with the variance determination.
  • variation in activity due to a single gene or a single genetic variance in a single gene is not sufficient to account for observed variation in patient response to a treatment, e.g., a drug, there are often other factors that account for some of the variation in patient response.
  • a treatment e.g., a drug
  • Haplotype is the cis a ⁇ angement of polymo ⁇ hic nucleotides on a particular chromosome.
  • Haplotype analysis has several advantages compared to the serial analysis of individual polymo ⁇ hisms at a locus with multiple polymo ⁇ hic sites.
  • haplotypes at a locus Of all the possible haplotypes at a locus (2 n haplotypes are theoretically possible at a locus with n binary polymo ⁇ hic sites) only a small fraction will generally occur at a significant frequency in human populations. Thus, association studies of haplotypes and phenotypes will involve testing fewer hypotheses. As a result there is a smaller probability of Type I e ⁇ ors, that is, false inferences that a particular variant is associated with a given phenotype. (2) The biological effect of each variance at a locus may be different both in magnitude and direction.
  • a polymo ⁇ hism in the 5' UTR may affect translational efficiency
  • a coding sequence polymo ⁇ hism may affect protein activity
  • a polymo ⁇ hism in the 3' UTR may affect mRNA folding and half life, and so on.
  • Haplotype analysis is the best method for assessing the interaction of variances at a locus.
  • haplotyping will be to identify the common haplotypes at selected loci that have multiple sites of variance. Haplotypes will usually be determined at the cDNA level. Two general approaches to identification of haplotyes will be employed. First, haplotypes will be infe ⁇ ed from the pattern of allele segregation in families collected by the Centre d'Etude Polymo ⁇ hisme Humaine. Cell lines from these families are available from the Coriell Repository. Cell lines for all members of families 884, 102, 104 and 1331 are cu ⁇ ently utilized. Cell lines from six additional families will also be used to increase the likelihood of detecting common haplotypes. This approach will be useful for cataloging common haplotypes and for validating methods on samples with known haplotypes.
  • haplotypes will be determined directly from cDNA using the T4E7 procedure.
  • T4E7 cleaves mismatched heteroduplex DNA at the site of the mismatch. If a heteroduplex contains only one mismatch, cleavage will result in the generation of two fragments. However, if a single heteroduplex (allele) contains two mismatches, cleavage will occur at two different sites resulting in the generation of three fragments. The appearance of a fragment whose size co ⁇ esponds to the distance between the two cleavage sites is diagnostic of the two mismatches being present on the same strand (allele).
  • T4E7 can be used to determine haplotypes in diploid cells.
  • An alternative method, allele specific PCR may be used for haplotyping.
  • PCR primers are designed to cover two sites of variance (either adjacent sites or sites spanning one or more internal variances). Two versions of each primer are synthesized, identical to each other except for the 3' terminal nucleotide. The 3' terminal nucleotide is designed so that it will hybridize to one but not the other variant base. PCR amplification is then attempted with all four possible primer combinations in separate wells.
  • a method of treatment is selected (and/or a method of administration) which co ⁇ elates positively with the particular variance presence or absence which provides the indication of effectiveness.
  • such selection can involve a variety of different choices, and the co ⁇ elation can involve a variety of different types of treatments, or choices of methods of treatment.
  • the selection may include choices between treatments or methods of administration where more than one method is likely to be effective, or where there is a range of expected effectiveness or different expected levels of contra-indication or deleterious effects.
  • the selection is preferably performed to select a treatment which will be as effective or more effective than other methods, while having a comparatively low level of deleterious effects.
  • a method is selected which has low such effects but which is expected to be effective in the patient.
  • the determination of the presence or absence of that at least one variance provides diagnostic methods, which can be used as indicated in the Summary above to select methods of treatment, methods of administration of a treatment, methods of selecting a patient or patients for a treatment, and others aspects in which the determination of the presence or absence of those variances provides useful information for selecting or designing or preparing methods or materials for medical use in the aspects of this invention.
  • variance determination or diagnostic methods can be performed in various ways as understood by those skilled in the art.
  • a sequence to be amplified includes at least one variance site, which is preferably a site or sites which provide variance information indicative of the effectiveness of a method of treatment or method of administration of a treatment, or effectiveness of a second method of treatment which reduces a deleterious effect of a first treatment method, or which enhances the effectiveness of a first method of treatment.
  • PCR polymerase chain reaction
  • the amplified sequence For convenient use of the amplified sequence, e.g., for sequencing, it is beneficial that the amplified sequence be of limited length, but still long enough to allow convenient and specific amplification. Thus, preferably the amplified sequence has a length as described in the Summary.
  • sequencing can utilize dye termination methods and mass spectrometric methods.
  • the sequencing generally involves a nucleic acid sequence which includes a variance site as indicated above in connection with amplification.
  • Such sequencing can directly provide determination of the presence or absence of a particular variance or set of variances, e.g., a haplotype, by inspection of the sequence (visually or by computer).
  • Such sequencing is generally conducted on PCR amplified sequences in order to provide sufficient signal for practical or reliable sequence determination.
  • probes can be of a variety of different types.
  • compositions Including Pharmaceutical Compositions Adapted to be Preferentially Effective in Patients Having Particular Genetic Characteristics
  • compositions which are adapted to be preferentially effective in patients who possess particular genetic characteristics i.e., in whom a particular variance or variances in one or more genes is present or absent (depending on whether the presence or the absence of the variance or variances in a patient is co ⁇ elated with an increased expectation of beneficial response).
  • the presence of a particular variance or variances may indicate that a patient can beneficially receive a significantly higher dosage of a drug than a patient having a different variance or variances.
  • the drug may be given for an indication that it may be used in the treatment of a particular disease or condition where the patient has at least one copy of a particular variance, variances, or variant form of a gene.
  • the regulatory agency may suggest use limited to particular groups or excluding particular groups or may state advantages of use or exclusion of such groups or may state a warning on the use of the drug in certain groups. Consistent with such suggestions and indications, such an agency may suggest or recommend the use of a diagnostic test to identify the presence or absence of the relevant variances in the prospective patient. Such diagnostic methods are described in this description.
  • this invention also includes drugs or pharmaceutical compositions which carry such a suggestion or statement of indication or warning or suggestion for a diagnostic test, and which may also be packaged with an insert or label stating the suggestion or indication or warning or suggestion for a diagnostic test.
  • an indication or suggestion can specify that a patient be heterozygous, or alternatively, homozygous for a particular variance or variances or variant form of a gene.
  • an indication or suggestion may specify that a patient have no more than one copy, or zero copies, of a particular variance, variances, or variant form of a gene.
  • a regulatory indication or suggestion may concern the variances or variant forms of a gene in normal cells of a patient and/or in cells involved in the disease or condition.
  • the response of the cancer cells can depend on the form of a gene remaining in cancer cells following loss of heterozygosity affecting that gene.
  • normal cells of the patient may contain a form of the gene which co ⁇ elates with effective treatment response, the absence of that form in cancer cells will mean that the treatment would be less likely to be effective in that patient than in another patient who retained in cancer cells the form of the gene which co ⁇ elated with effective treatment response.
  • a particular compound useful in this invention can be administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s).
  • a therapeutically effective amount of a agent or agents such as these is administered.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit large therapeutic indices are prefe ⁇ ed.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et. al., in The Pharmacological Basis of Therapeutics. 1975, Ch. 1 p.l). It should be noted that the attending physician would know how to and when to terminate, inte ⁇ upt, or adjust administration due to toxicity, or to organ dysfunctions.
  • the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administrated dose in the management of disorder of interest will vary with the severity of the condition to be treated and the route of admimstration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine. Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences.
  • Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal admimstration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penevers appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable ca ⁇ iers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are inco ⁇ orated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and or polyvinylpy ⁇ olidone (PVP).
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl
  • disintegrating agents may be added, such as the cross-linked polyvinyl py ⁇ olidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl py ⁇ olidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • 5-FU is a biologically inactive pyrimidine analog which must be phosphorylated and ribosylated to the nucleoside analog fluorodeoxyuridine monophosphate (FdUMP) to have clinical activity.
  • FdUMP formation can occur via several routes, summarized in Figure 1.
  • 5-FU may be converted by uridine phosphorylase to fluorouridine (FUdR; the reverse reaction is catalyzed by uridine nucleosidase) and then to fluorouridine monophosphate (FUMP) by uridine kinase, or FUMP may be formed from 5-FU in one step via transfer of a phosphoribosyl group from 5 -phosphoribosyl- 1-pyrophosphate (PRPP), catalyzed by orotate phosphoribosyl transferase.
  • FUMP can be converted to FUDP and subsequently
  • FUTP by a nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively.
  • FUTP is inco ⁇ orated into RNA by RNA polymerases. which may account in part for 5-FU toxicity as a result of effects on processing or function (e.g. translation).
  • FUDP may be reduced to the dinucleotide level, FdUDP (fluorodeoxyuridine diphosphate) by ribonucleotide diphosphate reductase, a heterodimeric enzyme.
  • FdUDP can then be converted to FdUTP by nucleoside diphosphate kinase and inco ⁇ orated into DNA by DNA polymerases which may account for some 5-FU toxicity.
  • Fluoropyrimidine modified DNA may also be targeted by the nucleotide excision repair process.
  • dUMP is the precursor of dTMP in de novo pyrimidine biosynthesis, a reaction catalyzed by thymidylate synthase and which consumes 5, 10-methylenetetrahydro folate, producing 7,8 dihydrofolate.
  • FdUMP forms an inhibitory (probably covalent) complex with thymidylate synthase in the presence of 5,10-methylenetetrahydro folate, thereby blocking formation of thymidylate (other than by the salvage pathway via thymidine kinase).
  • the complex anabolism of FdUMP can be simplified by giving the deoxyribonucleoside of 5-FU,
  • 5-fluorodeoxyuridine also called floxuridine; FUdR
  • FUdR 5-fluorodeoxyuridine
  • FdUMP in one step by thymidine kinase.
  • FUdR is also rapidly converted back to 5-FU by the bidirectional enzyme thymidine phophorylase.
  • Metabolic elimination of 5-FU occurs via a three step pathway leading to ⁇ - alanine.
  • the first and rate limiting enzyme in the elimination pathway is dihydropyrimidine dehydrogenase (DPD), which transforms more than 80% of a dose of 5-FU to the inactive dihydrofluorouracil form.
  • DPD dihydropyrimidine dehydrogenase
  • dihydropyrimidinase catalyzes opening of the pyrimidine ring to form 5-fluoro- ⁇ - ureidopropionate and then ⁇ -ureidopropionase (also called ⁇ -alanine synthase) catalyzes formation of 2-fluoro- ⁇ -alanine.
  • the first two reactions are reversible.
  • Intracellular reduced folate levels can potentiate 5-FU action by increasing 5,10-methylenetetrahydrofolate levels (5,10-methyleneTHF; see center of Figure 2), thereby stabilizing the ternary inhibitory complex formed with thymidylate synthase and FdUMP.
  • This is the basis for therapeutic modulation of 5-FU with FA.
  • conversion of folinic acid (5-formylTHF) to 5,10-methenylTHF the precursor of 5,10-methyleneTHF, requires methenyltetrahydrofolate synthetase (enzyme 2 in the Figure).
  • levels of 5,10-methyleneTHF may be affected directly by the activity of methyleneltetrahydro folate dehydrogenase, methylenkortrahydrofolate reductase, serine transhydroxymethylase and the glycine cleavage system enzymes (7, 8, 10 and 11 in Fig. 2), and indirectly by the other enzymes shown in the Figure.
  • Folate receptor 1 is a high affinity (nanomolar range) receptor for reduced folates.
  • Three restriction fragment length polymo ⁇ hisms (RFLPs) have been reported at the FR1 locus (Campbell et al., 1991).
  • Reduced folates are also transported by folate receptor gamma and by a low affinity (1 uM) folate transporter. 15-fold variation in levels of folate transporter have been described in unselected tumor cell lines (Moscow et al., 1997).
  • FdUMP, FUTP or FdUTP catabolism of 5-FU
  • depletion of cellular 5,10- methylenetetrahydrofolate may be causally related to variation in clinical effect of 5- FU FA.
  • genes not listed in the Table include DNA and RNA polymerases and DNA repair enzymes, some of which (e.g. DNA polymerase b and RNA polymerase II 220 and 33 kD subunits) have already been screened for polymo ⁇ hism.
  • DNA and RNA polymerases and DNA repair enzymes some of which (e.g. DNA polymerase b and RNA polymerase II 220 and 33 kD subunits) have already been screened for polymo ⁇ hism.
  • Those additional genes are also useful in the present invention.
  • For several potential candidate genes there are mammalian cDNAs in GenBank but no human cDNA. For example, there is a 1 ,420 nucleotide full length rat ⁇ -ureidopropionase cDNA.
  • Four overlapping human ESTs (F06711, H19181, Rl 1806 and W55897) span 691 nucleotides of the rat coding sequence with >90% nucleotide
  • genes related to modulation of the action of 5-FU FA have been analyzed for genetic variation; thymidylate synthase, ribonucleotide reductase (Ml subunit only), dihydrofolate reductase and dihydropyrimidine dehydrogenase cDNAs. 36 unrelated individuals were screened using 6 SSCP conditions and DNA sequencing. Other investigators have identified variances in MTHFR, methionine synthase and folate receptor. These findings are summarized in Table 3.
  • DPD dihydropynmidine dehydrogenase
  • Variances in the exemplary genes above which affect the activity of the co ⁇ esponding gene product have the potential to modulate the activity of 5-FU/FA and thereby provide predictive capability concerning the efficacy of such treatment in a particular patient.
  • predictive capability can further be provided by the joint determination of multiple variances, in one or a plurality of genes or both.
  • variances can provide such predictive capability for other treatments, e.g., treatments with other compounds, which involve these genes.
  • 5-fluorouracil is a widely used chemotherapy drug.
  • the effectiveness of 5-FU is potentiated by folinic acid (FA; generic name: leukovorin).
  • FA folinic acid
  • the combination of 5-FU and FA is standard therapy for stage III/TV colon cancer.
  • Patient responses to 5-FU and 5-FU FA vary widely, ranging from complete remission of cancer to severe toxicity.
  • 5-FU Clinical Use and Effectiveness of 5-FU and 5-FU/FA 5-FU is a pyrimidine analog in clinical use since 1957.
  • 5-FU is used in the standard treatment of gastrointestinal, breast and head and neck cancers. Clinical trials have also shown responses in cancer of the bladder, ovary, cervix, prostate and pancreas. The remainder of this discussion will concern colorectal cancer.
  • 5-FU is used both in the adjuvant therapy of Dukes Stage B and C cancer and in the treatment of disseminated cancer. 5-FU alone produces partial remissions in 10- 30% of advanced colorectal cancers, however only a few percent of patients have complete remissions, and no benefit in survival has been demonstrated.
  • 5-FU has been used in combination with PALA, a pyrimidine synthesis inhibitor, to deplete cellular pools of UTP and thereby enhance formation of FUTP; in combination with methotrexate, to inhibit purine anabolism, leading to increased PRPP levels and consequent increased conversion of 5-FU to its active nucleotide metabolites; and in combination with folinic acid, which increases intracellular pools of reduced folate, driving formation of the ternary inhibitory complex formed by 5,10 methylenetetrahydrofolate, FdUMP and thymidylate synthase.
  • Levamisole, interferon and alkylating agents have also been used in combination with 5-FU.
  • 5- FU/Levamisole and 5-FU/FA are widely used in the adjuvant treatment of colon cancer, while 5-FU/FA is the most commonly used regimen for advanced colorectal cancer.
  • 5-FU/FA is the most commonly used regimen for advanced colorectal cancer.
  • Leukovorin folinic acid
  • 5-FU modulator a variety of other molecules have been used with 5-FU, including, for example, interferon-alpha, hydroxyurea, N-phosphonacetyl-L-aspartate, dipyridamole, levamisole, methotrexate, trimetrexate glucuronate, cisplatin and radiotherapy.
  • S-l is a novel oral anticancer drug, composed of the 5-FU prodrug tegafur plus gimestat (CDHP) and otastat potassium (Oxo) in a molar ratio of 1 :0.4: 1 , with CDHP inhibiting dihydropyrimidine dehydrogenase in order to prolong 5-FU concentrations in blood and tumour and Oxo present as a gastrointestinal protectant.
  • CDHP 5-FU prodrug tegafur plus gimestat
  • Oxo otastat potassium
  • 5-FU toxicity has been well documented in randomized clinical trials. Patients receiving 5-FU/FA are at even greater risk of toxic reactions and must be monitored carefully during therapy. A variety of side effects have been observed, affecting the gastrointestinal tract, bone ma ⁇ ow, heart and CNS. The most common toxic reactions are nausea and anorexia, which can be followed by life threatening mucositis, enteritis and dia ⁇ hea. Leukopenia is also a problem in some patients, particularly with the weekly dosage regimen. In a recent randomized trial of weekly vs. monthly 5-FU/FA, there were 7 deaths related to drug toxicity among 372 treated patients (1.9%; Buroker et al. 1994).
  • 5-FU is inactivated by the same metabolic pathway as thymine and uracil
  • DPD catalyzes the first, rate limiting step in pyrimidine catabolism and accounts for elimination of most 5-FU.
  • Normal individuals eliminate 5-FU with a half life of -10-15 minutes and excrete only 10% of a dose unchanged in the urine.
  • people genetically deficient in DPD eliminate 5-FU with a half life of
  • DPD deficiency has two climcal presentations: (i) an inborn e ⁇ or of metabolism causing some degree of neurologic dysfunction or (ii) asymtomatic until revealed by exposure to 5-FU or other pyrimidine analogs. With either presentation there is combined hyperuraciluria and hyperthyminuria. The vastly increased 5-FU half life in DPD deficient individuals causes severe toxicity and even death. Recently several mutations have been identified in DPD genes of deficient individuals (Wei et al., 1996), however none of these alleles appears to occur at appreciable frequency, so the cause of wide population variation in DPD levels is still not understood.
  • DPD Dihydropyrimidine dehydrogenase
  • DPD dihydropyrimidine dehydrogenase
  • DPD levels Correlate with Response to 5-FU Intratumoral DPD levels have been measured in patients receiving 5-FU chemotherapy. When complete responders were compared to partial or nonresponders, DPD levels were lower in the compete responders (Etienne et al., 1995). Leukocyte DPD levels have also been measured in patients receiving 5- FU/FA chemotherapy. When patients were divided into 3 groups: high, medium and low DPD activity, the frequency of serious side effects was highest in the low DPD group and vice versa (Katona et al., 1997).
  • DPD cDNAs have been cloned from a variety of higher eukaryotes and binding sites for its cofactors, prosthetic groups and substrate have been defined experimentally or by analogy with known consensus motifs (Yokata et al., 1994).
  • the DPD polymo ⁇ hisms that affect protein sequence occur at amino acids 29 (cys/arg) and 166 (met val) in the amino-terminal one-third of the protein.
  • Phylogenetic comparison of this region from boar, human, cow, fly, and bacteria shows that there are actually two highly conserved motifs that resemble either iron sulfur or zinc binding motifs, the latter being more likely due to the spacing of the cysteine residues.
  • the region around the met/val polymo ⁇ hism at amino acid 166 is highly conserved. Even the spacing of the putative zinc-finger domains is maintained between distantly related species, hinting at their importance. Since amino acid 166 is close to a highly conserved (and probably functionally important) region and is itself conserved, being a methionine in all species, it seems likely that perturbations in this position would have consequence.
  • the polymo ⁇ hism substitutes a long amino acid side chain capable of hydrogen bonding (methionine) for a compact, hydrophobic amino acid (valine). The region around amino acid 29 is not as well conserved.
  • GenBank numbers There are 24 genes in the Table, four of which we have already surveyed for polymo ⁇ hism (italicized genes). The genes with GenBank numbers are cu ⁇ ently being screened for variances. Genes lacking GenBank numbers are not yet represented in GenBank as full length cDNAs; but will be scanned using relevant EST collections or using sequences from other publicly available sources.
  • Example 6 Drugs Targeting Genes Involved in Folate Transport and Metabolism
  • Table 6 identifies certain drug classes used for treatment of identified disorders, along with a brief characterization of the action of the drug.
  • Exemplary drugs are identified within the individual classes. Variable response of patients to administration of drugs of these classes, or administration of the specific drugs can be used in identifying variances responsible for such variable response. As described above, those variances can then be used in diagnostic tests, methods of selecting a treatment, methods of treating a patient, or other methods utilizing genetic variance information as otherwise described.
  • Drugs which affect or are affected by folate metabolism A wide spectrum of diseases are treated with drugs that affect folate metabolism. Some drugs are used in the treatment of several diseases. All of the listed drugs are frequently used in combination with other drugs. For example methotrexate is used in cancer chemotherapy with cytoxan and fluoruracil to treat breast cancer, among other combinations.
  • the new folate analogs include quinazoline derivatives such as ZD1694 (Tomudex, AstraZeneca) which requires Reduced Folate Carrier (RFC) mediated cell uptake and polyglutamation by Folylpolyglutamate Synthetase (FPGS); ZD9331 (AstraZeneca), which requires the RFC but is not polyglutamated by FPGS; LY231514 (Eli Lilly Research Labs, Indianapolis, IN) is a multi targeted py ⁇ olopyrimidine analogue antifolate which requires the RFC and polyglutamation; GW1843 (1843U89, Glaxo Wellcome) is a benzoquinazoline compound with potent TS inhibitory activity, and which enters cells via the RFC but is polyglutamated only to the diglutamate, which leads to higher cellular retention without augmenting TS inhibitory activity; AG337 (p.o.
  • AG331 both by Agouron, La Jolla, CA, now part of Warner Lambert
  • AG331 lipophilic TS inhibitors with action independent of the RFC and polyglutamation by FPGS
  • trimetrexate US Bioscience
  • Aminopterin is an older drug which has received renewed attention recently
  • edatrexate, piritrexim and lometrexol are other antifolate drugs.
  • 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid are structures into which a variety of modifications have been introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain (see article below).
  • Lilly have recently synthesized a new series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates which are being evaluated both as antineoplastic and antiarthritic agents.
  • Homocysteine is a proven risk factor for cardiovascular disease.
  • One important role of the folate cofactor 5-methyltetrahydro folate is the provision of a methyl group for the remethylation of homocysteine to methionine by the enzyme methionine synthase.
  • Variation in the enzymes of folate metabolism for example methionine syntase or methylenetetrahydrofolate reductase (MTHFR), may affect the levels of 5-methyltetrahydrofolate or other folates that in turn influence homocysteine levels.
  • the contribution of elevated homocysteine to atherosclerosis, thromboembolic disease and other forms of vascular and heart disease may vary from one patient to another.
  • Such variation may be attributable, at least in part, to genetically determined variation in the levels or function of the enzymes of folate metabolism described in this application. Assistance of clinical development or use of drugs to treat said cardiovascular diseases might be afforded by an understanding of which patients are most likely to benefit. This is true whether the drugs are aimed at the modulation of folate levels (e.g. supplemental folate) or at other known causes of cardiovascular disease (e.g. lipid lowering drugs such as statins, or antithrombotic drugs such as salicylates, heparin or GPIIIa/IIb inhibitors).
  • lipid lowering drugs such as statins, or antithrombotic drugs such as salicylates, heparin or GPIIIa/IIb inhibitors.
  • phencyclidine an NMDA receptor antagonist
  • NMDA receptor function closely resembling schizophrenia in normal individuals
  • the amino acid glycine is an obligatory coagonist (with glutamate) at NMDA receptors (via its action at a strychnine-insensitive binding site on the NMDA receptor complex), and consequently glycine or glycinergic agents (e.g. glycine, the glycine receptor partial agonist, D-cycloserine, or the glycine prodrug milacemide) have been tried as an adjunct to conventional antipsychotics for the treatment of schizophrenia.
  • glycine, the glycine receptor partial agonist, D-cycloserine, or the glycine prodrug milacemide have been tried as an adjunct to conventional antipsychotics for the treatment of schizophrenia.
  • Several trials have demonstrated a moderate improvement in negative symptoms of schizophrenia.
  • the endogenous levels of glycine in neurons may affect the response to glycine or glycinergic drugs.
  • inte ⁇ atient variation in glycine metabolism may affect drug efficacy.
  • genes involved in the related pathways of pyrimidine transport and metabolism are useful in the aspects of the present invention, e.g., for identifying variances responsible for variable treatment response, diagnostic methods, and methods of selecting a patient to receive a treatment.
  • Exemplary genes are provided below and are further identified by cellular function. Genes involved in those functions are generally useful in the present invention.
  • Table 9 Genes affecting the action of drugs which modulate pyrimidine metabolism.
  • a variety of proliferative diseases, especially cancer, are treated with drugs that affect pyrimidine metabolism. All of the listed drugs are frequently used in combination with other drugs.
  • pyrimidine analogs in clinical development for a wide variety of indications.
  • One of the most common indications is cancer and leukemia and lymphoma of various types.
  • 2',2'-difluorodeoxycytidine (gemcitabine; Gemzar) is a pyrimidine nucleoside drug with clinical efficacy in several common solid cancers
  • cytosine arabinoside (ARA-C) is another pyrimidine analog used in the treatment of leukemia
  • 2-chlorodeoxyadenosine and fludarabine F-araA are also used as antineoplastic drugs.
  • 2'-deoxy-2'-(fluoromethylene) cytidine (MDL 101,731, Kyowa Hakko Kogyo Co.), 2',2'-difluorodeoxycytidine, 5- aza-2'deoxycytidine (decitabine), 5-azacytidine, 5-azadeoxycytidine, and are under development as antineoplastic drugs.
  • CNS Drugs - Pyrimidine Pathway The pyrimidine nucleoside, uridine, has been proposed as a potential supplement in the treatment of psychosis based on its ability to reduce haloperidol- induced dopamine release.
  • uridine pyrimidine nucleoside
  • haloperidol might enhance the antipsychotic action of standard neuroleptics, allowing for a reduction in dose and thereby a reduction in the frequency of side effects.
  • the presumed mechanism is interaction with dopamine or GABA neurotransmission.
  • pyrimidine transporters or pyrimidine de novo or salvage biosynthetic enzymes, or pyrimidine catabolic enzymes may affect the action of neuroleptics, or their modulation by pyrimidine nucleosides or pyrimidine analogs.
  • Uridine triphosphate, uridine diphosphate, cytidine triphosphate, and deoxythymidine triphosphate all induce concentration-dependent increases in the release of thromboxane A2 from cultured glial cells, indicating a possible role in brain response to damage in vivo.
  • Other cancers such as head and neck, breast, pancreas, other gastrointestinal cancers including stomach and intestinal may be directly targeted by therapeutic intervention that affects DNA methylation levels, pyrimidine synthesis, transport, and degradation pathways.
  • Many neurological diseases in both the CNS and the periphery may also be affected by therapeutic intervention of DNA methylation, pyrimidine synthesis, transport, and degradation pathways. Such intervention may be of therapeutic benefit to halt, retard, and or reduce symptoms of these often debilitating diseases.
  • 5-FU prodrugs drugs that affect DNA methylation pathways
  • other drugs that have been developed for similar indications as 5-FU.
  • 5-FU prodrugs are generally modified fluoropyrimidines that require one or more enzymatic activation steps for conversion into 5-FU.
  • the activation steps may result in prolonged drug half-life and/or selective drug activation (i.e. conversion to 5-FU) in tumor cells.
  • Examples of such drugs include capecitabine (Xeloda, Roche), a drug that is converted to 5-FU by a three-step pathway involving Carboxylesterase 1, Cytidine Deaminase and Thymidine Phosphorylase.
  • Another 5-FU prodrug is 5'deoxy 5-FU (Furtulon, Roche) which is converted to 5-FU by Thymidine Phosphorylase and or Uridine Phosphorylase.
  • Another 5-FU prodrug is l-(tetrahydro-2-furanyl)-5- fluorouracil (FT, ftorafur, Tegafur, Taiho - Bristol Myers Squibb), a prodrug that is converted to 5-FU by cytochrome P450 enzyme, CYP3A4.
  • He ⁇ es virus thymidine kinase phosphorylates many 5 -substituted 2'- deoxyuridines, analogs of thymidine (e.g., idoxuridine, trifluridine, edoxudine, brivudine) and 5-substituted arabinofuranosyluracil derivatives (e.g., 5-*Et-Ara-U, BV-Ara-U, Cl-Ara-U).
  • the 5'-monophosphates are further phosphorylated by cellular enzymes to the 5'-triphosphates, which are usually competitive inhibitors of the viral-coded DNA polymerases.
  • retroviruses including but not limited to human immunodeficiency viruses do not encode specific enzymes required for the metabolism of the purine or pyrimidine nucleotides to their co ⁇ esponding 5'- triphosphates. Therefore, 2',3'-dideoxynucleosides and acyclic nucleoside phosphonates must be phosphorylated and metabolized by host cell kinases and other enzymes of purine and/or pyrimidine metabolism. In this way, affecting the pyrimidine synthetic, transport, or degradation pathways by candidate therapeutic intervention may be therapeutic beneficial in treating retroviral infections.
  • Excamples of candidate antivirals that may be affected by alteration of pyrimidine synthetic, transport, or degradation pathwyas are azidothymidine (AZT), acyclovir, and ganciclovir. These and other drugs have been used both as antivirals and antineoplastic agents.
  • drugs with activity against cancers usually treated with regimens containing 5-FU include Suramin, a bis- hexasulfonated napthylurea; 6-hydroxymethylacylfulvene (HMAF; MGI 114); LY295501; bizelesin (U-7779; NSC615291), ONYX-015, monoclonal antibodies (e.g. 17-1 A and MN-14), protein synthesis inhibitors such as RA 700, and angiogenesis inhibitors such as PF 4.
  • Still other drugs may prevent colorectal cancer by preventing the formation of colorectal polyps (eg, cyclooxygenase inhibitors may induce apoptosis of polyps).
  • PROTOCOL TITLE Case-control study to determine the relationship between toxicity of 5-fluorouracil (5-FU) given with folinic acid (FA) to patients with solid tumors and DNA sequence variances in enzymes that mediate the action of 5-FU and FA.
  • ECG Electrocardiogram e.g. For example o Degrees Fahrenheit
  • the primary objective of this study is to compare the variance frequency distribution in the dihydropyrimidine dehydrogenase (DPD) gene between two groups of patients with solid tumors, treated by weekly or monthly regimen of 5-FU+FA and defined by level of toxicity (graded according to the NCI common toxicity criteria) as:
  • the secondary objectives of the study are to determine the DPD gene haplotype frequency distribution and the variance and/or haplotype frequency distributions in selected genes (other than DPD gene) between two groups of patients with solid tumors, treated by weekly or monthly regimen of 5-FU+FA and defined by level of toxicity. Analyses will be done globally, then by regimen (monthly vs. weekly) and by type of toxicity (gastrointestinal vs. bone marrow).
  • Study Population Patients treated with 5-FU+FA for solid tumors at the Massachusetts General Hospital, Dana-Farber Cancer Institute and Brigham and Women's Hospital.
  • StudyGroups Patients will be divided into two groups depending on the degree of toxicity they experienced with treatment, if any: patients with high toxicity (grade III / IV on NCI criteria), patients with minimal toxicity (grade 0 / 1 / II on NCI criteria)
  • Visit Schedule One visit to sign the informed consent form and to collect blood sample.
  • Chemotherapy of cancer involves use of highly toxic drugs with na ⁇ ow therapeutic indices. Although progress has been made in the chemotherapeutic treatment of selected malignancies, most adult solid cancers remain highly refractory to treatment. Nonetheless, chemotherapy is the standard of care for most disseminated solid cancers. Chemotherapy often results in a significant fraction of treated patients suffering unpleasant or life-threatening side effects while receiving little or no clinical benefit; other patients may suffer few side effects and/or have complete remission or even cure. Any test that could predict response to chemotherapy, even partially, would allow more selective use of toxic drugs, and could thereby significantly improve efficacy of oncologic drug use, with the potential to both reduce side effects and increase the fraction of responders.
  • Chemotherapy is also expensive, not just because the drugs are often costly, but also because administering highly toxic drugs requires close monitoring by carefully trained personnel, and because hospitalization is often required for treatment of (or monitoring for) toxic drug reactions. Information that would allow patients to be divided into likely responder vs. non-responder (or likely side effect) groups, only the former to receive treatment, would therefore also have a significant impact on the economics of cancer drug use.
  • TPMT testing is done using an enzyme assay, however the TPMT gene has been cloned and mutations associated with low TPMT levels have been identified; genetic testing is beginning to supplant enzyme assays because genetic tests are more easily standardized and economical.
  • 5-FU is a pyrimidine analog in clinical use since 1957.
  • 5-FU is used in the standard treatment of gastrointestinal, breast and head and neck cancers.
  • Clinical trials have also shown responses in cancer of the bladder, ovary, cervix, prostate and pancreas. The remainder of this discussion will concern colorectal cancer.
  • 5-FU is used both in the adjuvant therapy of Dukes Stage B and C cancer and in the treatment of disseminated cancer.
  • 5-FU alone produces partial remissions in 10 - 30% of advanced colorectal cancers, however only a few percent of patients have complete remissions.
  • a variety of biochemically motivated strategies for modulating 5-FU activity have been tested.
  • 5-FU has been used in combination with PALA, a pyrimidine synthesis inhibitor, to deplete cellular pools of UTP and thereby enhance formation of FUTP; in combination with methotrexate, to inhibit purine anabolism, leading to increased PRPP levels and consequent increased conversion of 5-FU to its active nucleotide metabolites; and in combination with folinic acid, which increases intracellular pools of reduced folate, driving formation of the ternary inhibitory complex formed by 5,10 methylenetetrahydrofolate, FdUMP and thymidylate synthase.
  • Levamisole, interferon and alkylating agents have also been used in combination with 5-FU.
  • 5- FU/Levamisole and 5-FU/FA are widely used in the adjuvant treatment of colon cancer, while 5-FU/FA is the most commonly used regimen for advanced colorectal cancer.
  • 5-FU/FA is the most commonly used regimen for advanced colorectal cancer.
  • Two major dosing regimens are used: 5-FU plus low dose FA given for five consecutive days followed by a 23 day interval, or once weekly bolus IV 5-FU plus high dose FA.
  • the higher FA dose results in plasma FA concentrations of 1 to 10 uM, comparable to those required for optimal 5-FU/FA synergy in tissue culture, however low dose FA (20 mg/m 2 vs.
  • 5-FU toxicity has been well documented in randomized climcal trials. Patients receiving 5-FU/FA are at even greater risk of toxic reactions and must be monitored carefully during therapy. A variety of side effects have been observed, affecting the gastrointestinal tract, bone ma ⁇ ow, heart and CNS. The most common toxic reactions are nausea and anorexia, which can be followed by life threatening mucositis, enteritis and dia ⁇ hea. Leukopenia is also a problem in some patients, particularly with the weekly dosage regimen. In a recent randomized trial of weekly vs. monthly 5-FU/FA there were 7 deaths related to drug toxicity among 372 treated patients (1.9%; Buroker et al. 1994).
  • FIG. 1 5-FU metabolism and inhibition of thymidylate formation.
  • Enzymes 1. uridine phosphorylase; 2. thymidine phosphorylase; 3. orotate phosphoribosyl transferase; 4. thymidine kinase; 5. uridine kinase; 6. ribonucleotide reductase; 7. thymidylate synthase; 8. dCMP deaminase; 9. nucleoside monophosphate kinase; 10. nucleoside diphosphate kinase; 11. nucleoside diphosphatase or cytidylate kinase; 12: thymine phosphorylase.
  • FH2 dihydrofolate
  • FH4 tetrahydrofolate.
  • the Figure is adapted from Goodman & Gilman's The Pharmacological Basis of Therapeutics, ninth edition, McGraw Hill, 1996, p. 1249.
  • 5-FU is a biologically inactive pyrimidine analog, which must be phosphorylated, and ribosylated to the nucleoside analog fluorodeoxyuridine monophosphate (FdUMP) to have clinical activity.
  • FdUMP formation can occur via several routes, summarized in Figure 1.
  • 5-FU may be converted by uridine phosphorylase to fluorouridine (FUdR; the reverse reaction is catalyzed by uridine nucleosidase) and then to fluorouridine monophosphate (FUMP) by uridine kinase, or FUMP may be formed from 5-FU in one step via transfer of a phosphoribosyl group from 5- phosphoribosyl-1-pyrophosphate (PRPP), catalyzed by orotate phosphoribosyl transferase.
  • PRPP 5- phosphoribosyl-1-pyrophosphate
  • FUMP can be converted to FUDP and subsequently FUTP by a nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively.
  • FUTP is inco ⁇ orated into RNA by RNA polymerases, which may account in part for 5-FU toxicity as a result of effects on processing or function (e.g. translation).
  • FUDP may be reduced to the dinucleotide level, FdUDP (fluorodeoxyuridine diphosphate) by ribonucleotide diphosphate reductase, a heterodimeric enzyme.
  • FdUDP can then be converted to FdUTP by nucleoside diphosphate kinase and inco ⁇ orated into DNA by DNA polymerases, which may account for some 5-FU toxicity.
  • Fluoropyrimidine modified DNA may also be targeted by the nucleotide excision repair process.
  • dUMP is the precursor of dTMP in de novo pyrimidine biosynthesis, a reaction catalyzed by thymidylate synthase and which consumes 5,10-methylenetetrahydro folate, producing 7,8 dihydrofolate.
  • FdUMP forms an inhibitory (probably covalent) complex with thymidylate synthase in the presence of 5,10-methylenetetrahydro folate, thereby 1) locking formation of thymidylate (other than by the salvage pathway via thymidine kinase).
  • the complex anabolism of FdUMP can be simplified by giving the deoxyribonucleoside of 5-FU, 5-fluorodeoxyuridine (also called floxuridine; FUdR), which can be converted to FdUMP in one step by thymidine kinase. However, FUdR is also rapidly converted back to 5-FU by the bi-directional enzyme thymidine phosphorylase.
  • Metabolic elimination of 5-FU occurs via a three-step pathway leading to -alanine.
  • the first and rate limiting enzyme in the elimination pathway is dihydropyrimidine dehydrogenase (DPD), which transforms more than 80% of a dose of 5-FU to the inactive dihydrofluorouracil form.
  • DPD dihydropyrimidine dehydrogenase
  • dihydropyrimidinase catalyzes opening of the pyrimidine ring to form 5-fluoro ⁇ ureidopropionate and then - ureidopropionase (also called -alanine synthase) catalyzes formation of 2-fluoro-- alanine.
  • the first two reactions are reversible.
  • Enzymes 1. Formimino-tetrahydrofolate cyclodeaminase; 2. methenyltetrahydrofolate synthetase; 3. methenyltetrahydrofolate cyclohydrolase; 4. formyltetrahydrofolate synthetase; 5. formyltetrahydrofolate hydrolase; 6. formyltetrahydrofolate dehydrogenase; 7. methylenetetrahydrofolate dehydrogenase; 8. methylenetetrahydrofolate reductase (MTHFR); 9. homocysteine methyltransferase (also called methionine synthetase); 10. serine transhydroxymethylase; 11.
  • THF tetrahydrofolate
  • DHF dihydrofolate
  • Intracellular reduced folate levels can potentiate 5-FU action by increasing 5,10- methyl-enetetrahydrofolate levels (5,10-methyleneTHF; see center of Figure 2), thereby stabilizing the ternary inhibitory complex formed with thymidylate synthase and FdUMP. This is the basis for therapeutic modulation of 5-FU with FA.
  • conversion of folinic acid (5-formylTHF) to 5,10-methenylTHF the precursor of 5,10-methyleneTHF, requires methenyltetrahydrofolate synthetase (enzyme 2 in the Figure).
  • levels of 5,10-methyleneTHF may be affected directly by the activity of methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate reductase, serine transhydroxymethylase and the glycine cleavage system enzymes (7, 8, 10 and 11 in Fig. 2), and indirectly by the other enzymes shown in the Figure.
  • RFLPs restriction fragment length polymo ⁇ hisms
  • 5-FU is inactivated by the same metabolic pathway as thymine and uracil (see above).
  • DPD catalyzes the first, rate-limiting step in pyrimidine catabolism and accounts for elimination of most 5-FU.
  • Normal individuals eliminate 5-FU with a half-life of -10-15 minutes and excrete only 10% of a dose unchanged in the urine.
  • people genetically deficient in DPD eliminate 5-FU with a half-life of -2.5 hours and excrete 90% of a dose unchanged in the urine (Diasio et al., 1988).
  • DPD deficiency has two clinical presentations: (i) an inborn e ⁇ or of metabolism causing some degree of neurologic dysfunction or (ii) asymptomatic until revealed by exposure to 5-FU or other pyrimidine analogs.
  • Intratumoral DPD levels have been measured in patients receiving 5-FU chemotherapy. When complete responders were compared to partial or non- responders, DPD levels were lower in the compete responders (Etienne et al., 1995). Leukocyte DPD levels has also been measured in patients receiving 5-FU/FA chemotherapy. When patients were divided into 3 groups: high, medium and low DPD activity, the frequency of serious side effects was highest in the low DPD group and vice versa (Katona et al., 1997).
  • Variagenics has aheady surveyed thymidylate synthase, ribonucleotide reductase (Ml subunit only), and dihydrofolate reductase and dihydropyrimidine dehydrogenase cDNAs for genetic variation. 36 unrelated individuals were screened using 6 SSCP conditions and DNA sequencing. Other investigators have identified variances in MTHFR, methionine synthase and folate receptor. These findings are summarized in Appendix I. XV. XVI. 2.1.5 Analysis of Haplotypes Increases Power of Genetic Analysis
  • the sequential testing for co ⁇ elation between phenotypes of interest and single nucleotide polymo ⁇ hisms may be adequate to detect associations if there are major effects associated with single nucleotide changes; certainly it is worth performing this type of analysis. However there is no way to know in advance whether there are major phenotypic effects associated with single nucleotide changes and, even if there are, there is no way to be sure that the salient variance has been identified by screening cDNAs. A more powerful way to address the question of genotype-phenotype co ⁇ elation is to assort genotypes into haplotypes.
  • Haplotype is the cis a ⁇ angement of polymo ⁇ hic nucleotides on a particular chromosome.
  • Haplotype analysis has several advantages compared to the serial analysis of individual polymo ⁇ hisms at a locus with multiple polymo ⁇ hic sites.
  • haplotypes at a locus Of all the possible haplotypes at a locus (2 n haplotypes are theoretically possible at a locus with n binary polymo ⁇ hic sites) only a small fraction will generally occur at a significant frequency in human populations. Thus, association studies of haplotypes and phenotypes will involve testing fewer hypotheses. As a result there is a smaller probability of Type I e ⁇ ors, that is, false inferences that a particular variant is associated with a given phenotype.
  • each variance at a locus may be different both in magnitude and direction.
  • a polymo ⁇ hism in the 5' UTR may affect translational efficiency
  • a coding sequence polymo ⁇ hism may affect protein activity
  • a polymo ⁇ hism in the 3' UTR may affect mRNA folding and half life, and so on.
  • Haplotype analysis is the best method for assessing the interaction of variances at a locus.
  • DPD cDNAs have been cloned from a variety of higher eukaryotes and binding sites for its cofactors, prosthetic groups and substrate have been defined experimentally or by analogy with known consensus motifs (Yokata et al., 1994).
  • the DPD polymo ⁇ hisms that affect protein sequence occur at amino acids 29 (cys/arg) and 166 (met val) in the amino-terminal one-third of the protein.
  • Phylogenetic comparison of this region from boar, human, cow, fly, and bacteria shows that there are actually two highly conserved motifs that resemble either iron sulfur or zinc binding motifs, the latter being more likely due to the spacing of the cysteine residues.
  • the region around the met/val polymo ⁇ hism at amino acid 166 is highly conserved. Even the spacing of the putative zinc-finger domains is maintained between distantly related species, hinting at their importance. Since amino acid 166 is close to a highly conserved (and probably functionally important) region and is itself conserved, being a methionine in all species, it seems likely that perturbations in this position would have consequence.
  • the polymo ⁇ hism substitutes a long amino acid side chain capable of hydrogen bonding (methionine) for a compact, hydrophobic amino acid (valine). The region around amino acid 29 is not as well conserved.
  • 5 -fluoro uracil is a fluorinated pyrimidine analog that is widely used in chemotherapy.
  • the effectiveness of 5-FU is potentiated by folinic acid (FA: generic name: leukovorin).
  • FA generic name: leukovorin
  • the combination of 5-FU and FA is standard therapy for stage III/IV colon cancer. Patient responses to 5-FU and 5-FU/FA vary widely, ranging from complete remission of cancer to severe toxicity.
  • DPD Dihydropyrimidine dehydrogenase
  • Total DPD deficiency (familial pyrimidinemia and pyridinuria) is a rare syndrome associated with 5-FU induced toxicity. A milder defect in DPD activity appears to account for the severe side effects that occur in l%-3% of unselected cancer patients (Milano and Etienne, 1994).
  • 5-FU and FA The major toxic manifestations of 5-FU and FA depend on the schedule of administration and occur mainly in rapidly dividing tissues such as bone ma ⁇ ow and the mucosal lining of the gastrointestinal tract.
  • This study is designed to test whether genetically encoded biochemical variations in the enzymes of pyrimidine catabolism, nucleotide metabolism and folic acid metabolism, among patients treated with a weekly or monthly schedule of 5-FU+FA, account for some of the variation in drug toxicity.
  • Applications of a successful pharmacogenetic study lie in the direction of safer, more efficacious, and hence more economical use of 5-FU, guided by genetic tests.
  • the primary objective of this study is to compare the variance frequency distribution in the dihydropyrimidine dehydrogenase (DPD) gene between two groups of patients with solid tumors, treated by weekly or monthly regimen of 5- FU+FA and defined by level of toxicity (graded according to the NCI common toxicity criteria) as:
  • the secondary objectives of the study are to determine the DPD gene haplotype frequency distribution and the variance and/or haplotype frequency distributions in selected genes (other than DPD gene -see Appendix I-) between two groups of patients with solid tumors, treated by weekly or monthly regimen of 5-FU+FA and defined by level of toxicity. Analyses will be done globally, then by regimen (monthly vs. weekly) and by type of toxicity (gastrointestinal vs. bone ma ⁇ ow).
  • the study will be done at selected medical institution.
  • the study is a single-center, case-control study.
  • the duration of the study is expected to be not more than 8 months.
  • Group 1 patients with high toxicity (grade III / IV on NCI criteria)
  • Group 2 patients with minimal toxicity (grade 0 / 1 / II on NCI criteria)
  • the primary endpoint of this study is to measure and compare genotype distributions of the DPD gene in patients with and without 5- FU+FA toxicity.
  • nucleotide 3925 44 patients per group
  • nucleotide 3937 43 patients per group.
  • a total of 90 patients (45 per group) will so be recruited.
  • the primary objective of this study is to compare the variance frequency distributions in the dihydropyrimidine dehydrogenase (DPD) gene between two groups of patients with solid tumors, treated by weekly or monthly regimen of 5- FU+FA and defined by level of toxicity (grade 0/1/11 vs. grade III/IV).
  • DPD dihydropyrimidine dehydrogenase
  • haplotype frequencies of r predetermined haplotypes We will also compare haplotype frequencies of r predetermined haplotypes.
  • the method of cladograms (Templeton et al., 1987) will be used in an attempt to find out the smallest possible number r.
  • the evolutionary relationships between present day haplotypes are represented as a tree or cladogram.
  • testing for unequal haplotype frequencies between cases and controls can be considered in the same framework as testing for unequal variance frequencies since a single variance can be considered as a haplotype of a single locus.
  • the relevant likelihood ratio test compares a model where two separate sets of haplotype frequencies apply to the cases and controls, to one where the entire sample is characterized by a single common set of haplotype frequencies. This can be performed by repeated use of a computer program (Terwilliger and Ott, 1994) to successively obtain the log-likelihood co ⁇ esponding to the set of haplotype frequency estimates on the cases (In L case ), on the controls (In L conlrol ) and on the overall (In L combiHed ).
  • the test statistic 2(ln L case + In L cmtrol - In L combin is then a chi- squared with r -1 degrees of freedom (where r is the number of haplotypes).
  • logistic regression will be used with case-control status as the outcome variable, and genotypes and covariates (plus possible interactions) as predictor variables.
  • An Advisory Committee will be formed to provide scientific and medical direction for the study and to oversee the administrative progress of the study.
  • the Advisory Committee will meet at least once a month to monitor subjects.
  • the Advisory Committee will determine whether the study should be stopped or amended for any reason.
  • the Advisory Committee will be comprised of the Director of Clinical Pharmacogenetics, Vice-President for Discovery Research from the study sponsor (and/or their designee) and participating investigators. The principal investigator will chair the Advisory Committee.
  • Protocol amendments must be submitted to the IRB/REB/EC. Protocol modifications that impact on subject safety, the scope of the investigation, or affect the scientific quality of the study must be approved by the IRB/REB EC and submitted to the appropriate regulatory authorities before initiation. However, Variagenics may, at any time, amend this protocol to eliminate an apparent immediate hazard to a subject. In this case, the appropriate regulatory authorities will be subsequently notified. In the event of a protocol modification, the subject consent form may require similar modifications.
  • the Principal Investigator must maintain the records of signed consent forms, CRFs, all co ⁇ espondences, dates of any monitoring visits, and records that support this information for a period of 15 years following notification by the study sponsor that the clinical investigations have been completed or discontinued. All local laws regarding retention of records must also be followed.
  • the IRB/REB/EC must be notified of completion or termination of the protocol. Within 3 months of protocol completion or termination, the investigator must provide a final clinical summary report to the IRB/REB/EC. The Principal Investigator must maintain an accurate and complete record of all submissions made to the IRB/REB EC, including a list of all reports and documents submitted. A copy of these reports should be sent to the study sponsor. XL VIII. REFERENCES
  • Folate binding protein is a marker for ovarian cancer. Cancer Reearch 51 : 5329-38.
  • Diasio RB Beavers TL, Ca ⁇ enter JT.(1988) Familial deficiency of dihydropyrimidine dehydrogenase. Biochemical basis for familial pyrimidinemia and severe 5-fluorouracil-induced toxicity. J Clin Invest 81 :47-51.
  • DPD Dihydropyrimidine dehydrogenase
  • lymphoblastoid cell lines should be satisfactory sources of nucleic acid for the genetic studies.
  • Evolution is the process of change and diversification of organisms through time, and evolutionary change affects mo ⁇ hology, physiology and reproduction of organisms, including humans. These evolutionary changes are the result of changes in the underlying genetic or hereditary material. Evolutionary changes in a group of interbreeding individuals or Mendelian population, or simply populations, are described in terms of changes in the frequency of genotypes and their constituent alleles. Genotype frequencies for any given generation is the result of the mating among members (genotypes) of their previous generation. Thus, the expected proportion of genotypes from a random union of individuals in a given population is essential for describing the total genetic variation for a population of any species.
  • the expected number of genotypes that could form from the random union of two alleles, A and a, of a gene are AA, Aa and aa.
  • the expected frequency of genotypes in a large, random mating population was discovered to remain constant from generation to generation; or achieve Hardy-Weinberg equilibrium, named after its discoverers.
  • the expected genotypic frequencies of alleles A and a are conventionally described in terms of p 2 + 2pq + q 2 in which p and q are the allele frequencies of A and a.
  • p is defined as the frequency of one allele and q as the frequency of another allele for a trait controlled by a pair of alleles (A and a).
  • p equals all of the alleles in individuals who are homozygous dominant (AA) and half of the alleles in individuals who are heterozygous (Aa) for this trait.
  • Linkage is the tendency of genes or DNA sequences (e.g. SNPs) to be inherited together as a consequence of their physical proximity on a single chromosome. The closer together the markers are, the lower the probability that they will be separated during DNA crossing over, and hence the greater the probability that they will be inherited together.
  • a mutational event introduces a "new" allele in the close proximity of a gene or an allele.
  • the new allele will tend to be inherited together with the alleles present on the "ancestral,” chromosome or haplotype.
  • linkage disequilibrium will decline over time due to recombination.
  • Linkage disequilibrium has been used to map disease genes. In general, both allele and haplotype frequencies differ among populations. Linkage disequilibrium is varied among the populations, being absent in some and highly significant in others.5
  • PlaR be the placebo response rate (0% ( PlaR ( 100%) and TntR be the treatment response rate (0% ( TntR ( 100%) of a classical clinical trial.
  • ObsRR is defined as the relative risk between TntR and PlaR:
  • ObsRR TntR / PlaR.
  • AAR, AaR, aaR as respectively the response rates of the AA, Aa and aa patients.
  • TntR AAR*p2 + AaR*2pq + aaR*q2.
  • a response rate equal to the placebo response rate (which means that the polymo ⁇ hism has no impact on natural disease evolution but only on drug action) and let's define ExpRR as the relative risk between AAR and aaR, as
  • TntR / PlaR (AAR*p2 + AaR*2pq + aaR*q2) / PlaR
  • max(ExpRR) The maximum of the expected relative risk, max(ExpRR), co ⁇ esponding to the case of heterozygous patients having the same response rate as the placebo rate, is such that:
  • haplotypes for this gene and (2p)(2p-l)/2 possible genotypes. And with 2 genes with pi and p2 SNP loci, we have [(2pl)(2pl- l)/2]*[(2p2)(2p2-l)/2] possibilities; and so on. Examining haplotypes instead of combinations of SNPs is especially useful when there is linkage disequilibrium enough to reduce the number of combinations to test, but not complete since in this latest case one SNP would be sufficient. Yet the problem of frequency above still remains with haplotypes instead of SNPs since the frequency of a haplotype cannot be higher than the highest SNP frequency involved.
  • the statistical significance of the difference between genotype frequencies associated to every variance can be assessed by a Pearson chi- squared test of homogeneity of proportions with 2 degrees of freedom, using the same Bonfe ⁇ oni's adjustment as above.
  • Testing for unequal haplotype frequencies between cases and controls can be considered in the same framework as testing for unequal variance frequencies since a single variance can be considered as a haplotype of a single locus.
  • the relevant likelihood ratio test compares a model where two seqarate sets of haplotype frequencies apply to the cases and controls, to one where the entire sample is characterized by a single common set of haplotype frequencies.
  • logistic regression can be used with case-control status as the outcome variable, and genotypes and covariates (plus possible interactions) as predictor variables.
  • Example 13 Method for Producing cDNA
  • Methods for producing cDNA are known to those skilled in the art, as are methods for amplifying and sequencing the cDNA or portions thereof.
  • An example of a useful cDNA production protocol is provided below. As recognized by those skilled in the art, other specific protocols can also be used.
  • This example describes the SSCP technique for identification of sequence variances of genes.
  • SSCP is usually paired with a DNA sequencing method, since the SSCP method does not provide the nucleotide identity of variances.
  • One useful sequencing method for example, is DNA cycle sequencing of 32 P labeled PCR products using the Femtomole DNA cycle sequencing kit from Promega (WI) and the instructions provided with the kit. Fragments are selected for DNA sequencing based on their behavior in the SSCP assay.
  • Single strand conformation polymo ⁇ hism screening is a widely used technique for identifying an discriminating DNA fragments which differ from each other by as little as a single nucleotide.
  • Orita et al. Detection of polymo ⁇ hisms of human DNA by gel electrophoresis as single-strand conformation polymo ⁇ hisms. Proc Natl Acad Sci US A. 86(8):2766-70, 1989
  • the technique was used on genomic DNA, however the same group showed that the technique works very well on PCR amplified DNA as well.
  • the technique has been used in hundreds of published papers, and modifications of the technique have been described in dozens of papers.
  • the enduring popularity of the technique is due to (1) a high degree of sensitivity to single base differences (>90%)
  • SSCP is almost always used together with DNA sequencing because SSCP does not directly provide the sequence basis of differential fragment mobility.
  • the basic steps of the SSCP procdure are described below.
  • the intent of SSCP screening is to identify a large number of gene variances it is useful to screen a relatively large number of individuals of different racial, ethnic and/or geographic origins. For example, 32 or 48 or 96 individuals is a convenient number to screen because gel electrophoresis apparatus are available with 96 wells (Applied Biosystems Division of Perkin Elmer Co ⁇ oration), allowing 3 X 32, 2 X 48 or 96 samples to be loaded per gel.
  • the 32 (or more) individuals screened should be representative of most of the worlds major populations. For example, an equal distribution of Africans, Europeans and Asians constitutes a reasonable screening set.
  • One useful source of cell lines from different populations is the Coriell Cell Repository (Camden, NJ), which sells EBV immortalized lyphoblastoid cells obtained from several thousand subjects, and includes the racial/ethnic/geographic background of cell line donors in its catalog.
  • a panel of cDNAs can be isolated from any specific target population.
  • SSCP can be used to analyze cDNAs or genomic DNAs. For many genes cDNA analysis is preferable because for many genes the full genomic sequence of the target gene is not available, however, this circumstance will change over the next few years.
  • RNA is isolated using an acid phenol protocol, sold in kit form as Trizol by Life Technologies (Gaithersberg, MD).
  • the unfractionated RNA is used to produce cDNA by the action of a modified Maloney Murine Leukemia Virus Reverse Transcriptase, purchased in kit form from Life Technologies (Superscript II kit).
  • the reverse transcriptase is primed with random hexamer primers to initiate cDNA synthesis along the whole length of the RNAs. This proved useful later in obtaining good PCR products from the 5' ends of some genes.
  • oligodT can be used to prime cDNA synthesis.
  • Material for SSCP analysis can be prepared by PCR amplification of the cDNA in the presence of one ⁇ 32 P labeled dNTP (usually ⁇ 32 P dCTP). Usually the concentration of nonradioactive dCTP is dropped from 200 uM (the standard concentration for each of the four dNTPs) to about 100 uM, and 32 P dCTP is added to a concentration of about 0.1-0.3 uM. This involves adding a 0.3- 1 ul (3-10 uCi) of 32 P cCTP to a 10 ul PCR reaction. Radioactive nucleotides can be purchased from DuPont/New England Nuclear.
  • the customary practice is to amplify about 200 base pair PCR products for SSCP, however, an alternative approach is to amplify about 0.8-1.4 kb fragments and then use several cocktails of restriction endonucleases to digest those into smaller fragments of about 0.1-0.4kb, aiming to have as many fragments as possible between .15 and .3 kb.
  • the digestion strategy has the advantage that less PCR is required, reducing both time and costs.
  • several different restriction enzyme digests can be performed on each set of samples (for example 96 cDNAs), and then each of the digests can be run separately on SSCP gels. This redundant method (where each nucleotide is surveyed in three different fragments) reduces both the false negative and false positive rates.
  • a site of variance might lie within 2 bases of the end of a fragment in one digest, and as a result not affect the conformation of that strand; the same variance, in a second or third digest, would likely lie in a location more prone to affect strand folding, and therefore be detected by SSCP.
  • the radiolabelled PCR products are diluted 1 :5 by adding formamide load buffer (80% formamide, IX SSCP gel buffer) and then denatured by heating to 90%C for 10 minutes, and then allowed to renature by quickly chilling on ice.
  • formamide load buffer 80% formamide, IX SSCP gel buffer
  • This procedure promotes intra- (rather than inter-) strand association and secondary structure formation.
  • the secondary structure of the single strands influences their mobility on nondenaturing gels, presumably by influencing the number of collisions between the molecule and the gel matrix (i.e., gel sieving). Even single base differences consistently produce changes in intrastrand folding sufficient to register as mobility differences on SSCP.
  • the single strands were then resolved on two gels, one a 5.5% acrylamide,
  • T4E7 T4 endonuclease VII
  • T4 endonuclease VII is derived from the bacteriophage T4.
  • T4 endonuclease VII is used by the bacteriophage to cleave branched DNA intermediates which form during replication so the DNA can be processed and packaged.
  • T4 endonuclease can also recognize and cleave heteroduplex DNA containing single base mismatches as well as deletions and insertions. This activity of the T4 endonuclease VII enzyme can be exploited to detect sequence variances present in the general population.

Abstract

L'invention concerne l'utilisation des informations sur la variance génétique en manière de transport de folates ou de gènes de métabolisme ou de transport de pyrimidine ou de gènes de métabolisme dans le traitement d'une maladie ou d'un état. Les informations sur la variance indiquent la réaction attendue d'un patient à un procédé de traitement déterminé. L'invention concerne également des procédés pour déterminer des informations pertinentes sur la variance et des procédés supplémentaires d'utilisation de ces informations concernant la variance.
EP99935759A 1998-07-20 1999-07-20 Variance de sequences de genes pouvant etre utile pour determiner le traitement d'une maladie Withdrawn EP1100964A1 (fr)

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GB9410620D0 (en) 1994-05-26 1994-07-13 Univ Mcgill cDNA for human mehylenetetrahydrofolate reductase
US6912492B1 (en) 1999-05-25 2005-06-28 University Of Medicine & Dentistry Of New Jersey Methods for diagnosing, preventing, and treating developmental disorders due to a combination of genetic and environmental factors
US6210950B1 (en) * 1999-05-25 2001-04-03 University Of Medicine And Dentistry Of New Jersey Methods for diagnosing, preventing, and treating developmental disorders due to a combination of genetic and environmental factors
WO2002000933A2 (fr) * 2000-06-23 2002-01-03 Interleukin Genetics, Inc. Essais de recherche systematique de modulateurs de la reaction inflammatoire ou immunitaire
JP2002153299A (ja) * 2000-11-22 2002-05-28 Sumikin Bioscience Kk ヒト補体活性化レクチンＲａＲＦの構成成分ＭＡＳＰ−１の遺伝子多型
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