WO2010025952A1 - Procédé de génotypage de variantes de région du promoteur ugt1a1*28, et systèmes à cet effet - Google Patents

Procédé de génotypage de variantes de région du promoteur ugt1a1*28, et systèmes à cet effet Download PDF

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WO2010025952A1
WO2010025952A1 PCT/EP2009/006479 EP2009006479W WO2010025952A1 WO 2010025952 A1 WO2010025952 A1 WO 2010025952A1 EP 2009006479 W EP2009006479 W EP 2009006479W WO 2010025952 A1 WO2010025952 A1 WO 2010025952A1
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ugt1a1
promoter region
genotyping
individual
drug
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PCT/EP2009/006479
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English (en)
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Tim O. Lankisch
Ursula Ehmer
Christian Strassburg
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Medizinische Hochschule Hannover
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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
    • 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

Definitions

  • the present invention relates to a method for genotyping an individual for (TA) 7 variant of the UGT1A1 promoter region.
  • the present invention relates to a specific method using the PCR TaqMan system for genotyping individuals for the presence of the (TA) ⁇ or (TA) 7 UGT1A1 genotype.
  • the present invention relates to a kit allowing genotyping of an individual for variants of the UGT1A1 promoter region as well as sys- terns for genotyping the same. Said kits and systems are particularly useful for the stratification of individuals undergoing drug therapy or being exposed to environmental occupational poison.
  • said methods, systems and kits are useful for prediction of safety, toxicity and/or efficacy of a substance, in particular, of a drug or product in drug therapy or of substances intended to be used as active ingredients in pharmaceutical or non- pharmaceutical applications.
  • the UDP-glucuronosyltransferase family of enzymes is a central metabolic system for the glucuronidation of hydrophobic endobiotic and xenobiotic compounds. Glucuronidation leads to the formation of water soluble metabolites which in the majority of cases results in an inactivation of the substrate and the subsequent elimination via bile or urine.
  • the spectrum of possible candidates for this pathway is broad and encompasses steroid hormones, bilirubin and bile acids as well as a vast array of therapeutic drugs, environmental organic substances including known human mutagens.
  • glucuronidation activities in the individual are a mechanism by which interindividual profiles of drug metabolism are believed to impact drug efficacy, drug side effects and the predisposition towards environmental mutagen-associated diseases such as cancer.
  • the human UGT1A proteins have been implicated as risk factors for both the development of cancer and unwanted drug side effects. This risk is determined by 3 differing features of the UGT1A gene locus.
  • the UGT1A gene locus (Genbank accession number AF297093) is regulated and expressed in a tissue specific fashion encompassing the hepatic isoforms UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9.
  • UGT1A and UGT2B genes are regulated in a polymorphic interindividual fashion leading to differing steady state levels of UGT mRNA, protein and enzymatic glucuronidation activity.
  • the molecular basis of this feature is presently not completely understood.
  • WO 99/57322 discloses various polymorphisms of the different UGT1A isoforms.
  • data of the genetic analysis and the analysis of the polymorphism are disclosed therein only. No information is provided with respect to the consequences of the various polymorphisms.
  • no information is given for an association of a specific polymorphism with a disease or disorder or enzy- matic activity.
  • Today various information have been published relating to a specific polymorphism of an UGT1A isoform which may lead to catalytically altered UGT1A protein variants which will be discussed in more detail below.
  • UGT1A1*28 is characterized by the insertion of a TA into the A(TA) 6 TAA element leading to A(TA) 7 TAA and a reduction of promoter activity to 30%.
  • This SNP is the genetic basis of Gilbert-Meulengracht's disease leading to unconjugated non-hemolytic hyperbilirubinemia because UGT1A1 is the only efficient metabolic pathway for the elimination of bilirubin from the human body.
  • UGT1A1*28 carrier status has been linked to the susceptibility towards breast cancer and the risk of unwanted intestinal side effects as well as myelotoxicity in colorectal cancer patients treated with irinotecan, a campto- thecin analog.
  • the Gilbert's syndrome is a common cause of mild jaundice which re- suits from unconjugated hyperbilirubinemia due to reduced glucuronidation of bilirubin by the UDP-glucuronosyltransferase 1A1 (UGT1A1). From the point of view of hepatology Gilbert's syndrome is considered benign because it does not lead to chronic liver dysfunction or fibrosis. However, recent reports have linked it to drug toxicity and cancer predisposition.
  • the genetic basis of Gilbert's syndrome in Caucasians is a homozygous TA insertion into the TATA box promoter region of the UGT1A1 gene (UGT1A1 * 28) on chromosome 2.
  • This variant exhibits 7 TA repeats (TA) 7 in- stead of the wild type 6 TA repeats (TA) 6 leading to a 70-80% reduction of bilirubin glucuronidation in homozygous UGT1A1 * 28 carriers.
  • This feature carries a high relevance because of its frequency amounting to approximately 0.4 in the Caucasian population, which would lead to 16% homozygous carriers (Bosma, P.J., J. Hepatol. 2003, 38(1): p. 107-17).
  • SNPs single nucleotide polymorphisms within the human UGT1A gene locus may also contribute to the phenotype of Gilbert's syndrome.
  • a G71 R exon variant with a frequency of approximately 0.23 (UGT1A1*6) has been described in Asians.
  • TA 8 polymorphisms, respectively, in homozygous or heterozygous individuals.
  • the TaqMan 5'-nuclease assay is an established method widely used e.g. for real time PCR quantification of viral loads in patients.
  • the design of genotype-specific probes permits a rapid allelic discrimination genotyping analysis.
  • TaqMan assays either have been published or are commercially available.
  • a method utilizing TaqMan PCR for allelic discrimination of the clinically most important variant - UGT1A1 * 28 - has not been described or published so far.
  • the present invention is generally concerned with the field of identifying an appropriate treatment regimen for a disease based upon a specific genotype of the individual to be treated. It is further concerned with a genetic basis of inter-patient variations in response to therapy, in particular drug therapy.
  • the present invention relates to a method for genotyping an indi- vidual for a (TA) 7 variant of the UGT1A1 promoter region known as UGT1A1 * 28. Said determination of a variant of said promoter region is useful in the field of therapeutics for optimising efficacy and safety of drug therapy as well as for allowing personalised medicine.
  • determining the genotype of an individual and, thus, determining said polymorphism may be useful during drug development processes and in guiding the optimal use of already approved compounds.
  • the present invention provides a method for genotyping an individual for a (TA) 7 variant of the UGT1A1 promoter region comprising the steps of: a) providing DNA of the individual to be tested; b) providing a pair of primer allowing amplification of the UGT1A1 promoter region containing the UGT1A1*28 polymorphism; c) providing probes of SEQ ID NOs. 1 and 2 or functional variants thereof which selectively hybridise to the UGT1A1 promoter region comprising marker moieties; d) amplification of the UGT1A1 promoter region containing the UGT1A1*28 polymorphism using Taq polymerase; e) detecting the UGT1AT28 genotype.
  • the inventors were successful in providing a method for accurate and cost effective genotyping of an individual based on PCR techniques, preferably the PCR TaqMan system.
  • Said method is based on using probes of SEQ ID NOs. 1 and 2 which selectively hybridise to the UGT1A1 promoter region comprising marker moieties and quencher moieties, optionally in combination with a minor groove binder (MGB), respectively, as well as functional variants thereof.
  • MGB minor groove binder
  • the term “functional variants thereof” refers to nucleotide sequences of SEQ ID NO. 1 or 2, respectively, varying at the 5' or 3'-end of said probe insofar that hybridisation may be optimised.
  • said probes of SEQ ID NOs are particularly, varying at the 5' or 3'-end of said probe insofar that hybridisation may be optimised.
  • the binding temperature of the probes is about 10 0 C higher than the binding temperature of the primers used for ampli- fying the UGT1A1 promoter region.
  • Diseases or conditions are commonly recognised in the art and designate the presence of signs and/or symptoms in an individual or patient that are generally recognised as abnormal.
  • Diseases or conditions may be diagnosed and categorised 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 result of diagnostic tests which may include, among others, laboratory tests.
  • Symptoms are subjective evidence of disease or patient condition, i.e. the patient's perception of an abnormal condition that differs from normal function, sensation, or experience, which may include, with limitation, physical disabilities, mobility, pain, and other changes from the normal condition experienced by an individual.
  • a beneficial change can, for example, include one or more of: the duration 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 dis- order.
  • drug or “prodrug” or “candidate ingredients of pharmaceutical compositions” as used herein refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to an individual 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, glycoproteins, lipoproteins, 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.
  • the chemical entity or the biological product may be a xenobiotic or an endogenous substance, preferably, the substance is a drug or prodrug.
  • drug may include, without limitation, compounds that are approved for sale as pharmaceutical products by government regulatory agencies (e.g., U.S. Food and Drug Administration (USFDA or FDA), European Medi- cines Evaluation Agency (EMEA), and a world regulatory body governing the International Conference of Harmonization (ICH) rules and guidelines), compounds that do not require approval by government regulatory agencies, food additives or supplements including compounds commonly characterized as vi- tamins, natural products, and completely or incompletely characterized mixtures of chemical entities including natural compounds or purified or partially purified natural products.
  • USFDA or FDA U.S. Food and Drug Administration
  • EMEA European Medi- cines Evaluation Agency
  • ICH International Conference of Harmonization
  • drug as used herein is synonymous with the terms "medicine”, “pharmaceutical product”, or “product”.
  • prodrug refers to a precursor of a drug, which is converted into the effective form, i.e. into the drug.
  • a "low molecular weight compound” has a molecular weight ⁇ 5,000 Da, more preferably ⁇ 2,500 Da, still more preferably ⁇ 1 ,000 Da, and most preferably ⁇ 700 Da.
  • the present invention relates in another aspect to a kit for genotyping an individual for a (TA) 7 variant of the UGT1A1 promoter region comprising: a) a pair of primer allowing amplification of the UGT1A1 promoter region containing the UGT1A1 *28 polymorphism; b) probes of SEQ ID NOs. 1 and 2 comprising marker moieties or functional fragments thereof; and c) optionally, means for amplification of the UGT1A1 promoter region containing the UGT1A1 * 28 polymorphism; d) optionally, instructions for genotyping the individual.
  • the present invention relates to a system for genotyping an individual for a (TA) 7 variant of the UGT1A1 promoter region comprising: a) a pair of primer allowing amplification of the UGT1A1 promoter region containing the UGT1A1 *28 polymorphism; b) probes of SEQ ID NOs. 1 and 2 comprising marker moieties or functional variants thereof; c) optionally, means for amplification of the UGT1A1 promoter region containing the UGT1A1 * 28 polymorphism; d) a measurement unit determining the amount of marker molecules; e) a processing unit for determining the UGT1A1 *28 genotype.
  • kit or system as well as the method described herein are particularly useful for predicting the safety, toxicity and/or efficacy of a substance, in par- ticular of a drug or product in a drug therapy or of candidate ingredients, e.g. chemical entities or biological entities identified as lead structures in drug research, of pharmaceutical or non-pharmaceutical compositions.
  • non-pharmaceutical composition refers to compositions used e.g. in the cosmetic field, or in the field of food, e.g. in the nutritional field.
  • Figure 1 Figure 1 provides a typical example of the allelic discrimination of the UGT1A1*28 variant by TaqMan PCR according to the present invention. NTC, no template control.
  • Figure 2 Figure 2 shows the analysis of 10 samples each of one wild type, one heterozygous, and one homozygous probe shows low inter- probe variability. Mean values for VIC and FAM fluorescence are shown for each probe with bars representing mean standard deviation.
  • Figure 3 Figure 3 is an analysis of 7 samples each in 7 different runs showing inter-assay variability demonstrating that a positive control with each run is preferred when conducting the method according to the present invention. Mean values for VIC and FAM fluorescence are shown for each probe with bars representing mean standard devia- tion. To ensure comparability fluorescence levels of NTC probes were subtracted from the probe levels.
  • the present invention relates to a method for genotyping an individual for a (TA) 7 variant of the UGT1A1 promoter region comprising the steps of: a) providing DNA of the individual to be tested; b) providing a pair of primer allowing amplification of the UGT1A1 promoter region containing the UGT1A1*28 polymorphism; c) providing probes of SEQ ID NOs. 1 and 2 or functional variants thereof which selectively hybridise to the UGT1A1 promoter region comprising marker moieties; d) amplification of the UGT1A1 promoter region containing the UGT1A1*28 polymorphism using Taq polymerase; e) detecting the UGT1A1*28 genotype.
  • the probes are particularly useful when applying the PCR TaqMan system.
  • the probes With the method according to the present invention it is possible to determine quantitatively the UGT1A1*28 genotype. That is, it is possible to determine the homo- zygotic or heterozygotic genotype of the promoter region in an individual.
  • the probes further comprise marker moieties.
  • said marker moieties are a fluorophore covalently attached to the 5'- end of the oligonucleotide probe, and a quencher at the 3'-end, or vice versa.
  • fluorophores fluorophores well-known in the art may be used, e.g. 6- carboxyfluorescein (FAM) or tetrachlorofluorescein (TET).
  • FAM 6- carboxyfluorescein
  • TET tetrachlorofluorescein
  • quencher moie- ties typical moieties as known in the art may be used, e.g. tetramethylrodamine (TAMRA) or dihydrocyclopyrroloindole tripeptide also calle a nonfluorescent quencher or NFQ.
  • TAMRA tetramethylrodamine
  • NFQ nonfluorescent quencher
  • MGB minor groove binder
  • the quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler's light source via fluorescence reso- nance energy transfer (FRET) as long as the fluorophore and the quencher in a proximity, quenching inhibits any fluorescence signals.
  • FRET fluorescence reso- nance energy transfer
  • the minor groove binder increase the melting temperature of the probe without increasing the probe length, thus, allowing the design of shorter probes.
  • the TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesises the nascent strand, the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probes releases a fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal Cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.
  • the probes according to the present invention in combination with the primer pair composed of the primers of SEQ ID NO. 3 and SEQ ID NO. 4 are particularly useful in the present invention. That is, said primers and probes are adjusted to allow optimal results in allelic discrimination of the UGT1A1 promoter region.
  • the difference in optimal binding temperature to the template should be 10 0 C between the primers and the probes. That is, when using functional variants of the primers the functional variants of the probes should be in the form to comply with the above requirement of having a difference of about 10 0 C for the binding temperature compared to the functional variants of the primer pair.
  • the first probe typically comprises a first fluorophore like FAM at the 5'-end while having a quencher at the 3'-end, preferably in combination with a MGB.
  • the second probe has a second fluorophore at its 5'- end, e.g. the fluorophore VIC® which is different to the fluorophore at the 5'- end of the first oligonucleotide probe while having a quencher molecule and, optionall, a MGB, at the 3'-end which may be identical with the quencher molecule and MGB at the 3'-end of the first probe.
  • the probes are composed of a oligonucleotide according to SEQ ID NO. 1 having FAM at the 5'-end and the non-fluorescent quencher/MGB at the 3'-end while the sec- ond probe of SEQ ID NO. 2 has a VIC® at the 5'-end and non-fluorescent quencher/MGB at the 3'-end.
  • the fluorophore may be reversed between the pair of probes.
  • the probe of SEQ ID NO. 1 is characterised in having 6 TA repeats while the probe according to SEQ ID NO. 2 has 7 TA repeats.
  • the probe of SEQ ID NO. 2 will selectively hybridise with the allelic variant of 7 TA repeats in the UGT1A1*28 promoter region while the probe according to SEQ ID NO. 1 will specifically hybridise with the allelic variation having 6 TA repeats.
  • the probes and primers as shown in Table 1 are used in the method according to the present invention.
  • the probes and primers are used in the concentration identified in Table 1.
  • the method according to the present invention allows to stratify individuals for the treatment of a disease, in particular, wherein the disease is cancer, neoplasia or chronic inflammatory disease, including inflammatory bowel disease, primary sclerosing cholangitis, and, in particular, wherein the disease is cancer.
  • 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 that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Often this dosage level is related to the peak or average serum levels resulting from administration of a drug at the particular dosage level.
  • a "frequency of administration” refers to how often in a speci- tied 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.
  • genomic DNA is used for the identification of said polymorphism.
  • the genomic DNA can be isolated from a sample of the individual's blood by known methods, e.g. by column chromatography and chemical processing. This genomic DNA, which represents the genotype of the individual to be investigated, forms the basis for the methods of the present invention.
  • the terms “effective” and “efficacy” includes both pharmacological efficacy and physiological safety.
  • Pharmacological efficacy 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 unstratified population.
  • Treatment may be ineffective 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.
  • 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 the polymorphism according to the present invention as identified above in cells of an individual to be treated.
  • the presence or absence of said variance is indicative that the treatment or method of admini- stration will be effective and safe in the individual.
  • the individual is selected for administration of the treatment.
  • the individual display the polymorphism of having a (TA) 7 independent of being homozygous or heterozygous, then it is necessary to decrease the dosage or to select a different therapeutic regimen for said individual.
  • the invention further provides a method for determining the predisposition to a physiological reaction of an individual to a chemical entity or a biologi- cal product which may be administered to an individual to treat or prevent or control a disease or condition.
  • physiological reaction refers to a reaction of in the individual. Said physiological reaction encompasses beneficial reactions and adverse reactions. Adverse reactions may be reactions resulting in side effects or may result in effects other than the in- tended beneficial effect.
  • the invention provides a kit for genotyping an individual for a (TA) 7 variant of the UGT1A1 promoter region comprising: a) a pair of primer allowing amplification of the UGT1A1 promoter region containing the UGT1A1 * 28 polymorphism; b) probes of SEQ ID NOs. 1 and 2 comprising marker moieties or functional fragments thereof; and c) optionally, means for amplification of the UGT1A1 promoter region containing the UGT1A1 *28 polymorphism; d) optionally, instructions for genotyping the individual.
  • the kit may also optionally contain other components, preferably other components adapted for identifying the UGT1A1 *28 polymorphism according to the present invention.
  • additional components can, for example, independently include a buffer or buffers, e.g., amplification buffers and hybridiza- tion buffers, which may be in liquid or dry form, a DNA polymerase, e.g., a polymerase suitable for carrying out PCR (e.g., a thermostable Taq DNA polymerase), and deoxy nucleotide triphosphates (dNTPs).
  • a buffer or buffers e.g., amplification buffers and hybridiza- tion buffers, which may be in liquid or dry form
  • a DNA polymerase e.g., a polymerase suitable for carrying out PCR (e.g., a thermostable Taq DNA polymerase)
  • dNTPs deoxy nucleotide triphosphates
  • the kit includes a nucleic acid or poly
  • the kit may be in form of an array or test arrangement which may, for example, in- elude a plurality of different antibodies, and/or a plurality of different nucleic acid sequences.
  • Sites in the array can allow capture and/or detection of nucleic acid sequences or gene products corresponding to different variances in one or more different genes including the polymorphism according to the present invention.
  • the array is arranged to provide variance detection for a plurality of variances in one or more genes which correlate with the effectiveness of one or more treatments of one or more diseases.
  • the kit may also optionally contain instructions for use.
  • the present invention provides a kit comprising the genetic detection reagents necessary for at least allowing allelic discrimination of the UGT1A1*28 promoter region.
  • the kit according to the present invention allows for conducting the method according to the present invention.
  • the invention also includes the use of such a kit or system to determine the genotype of one or more individuals with respect to the UGT1AT28 poly- morphsim. Such use can include providing a result or report indicating the presence and/or absence of one or more variant forms of UGT1A1 promoter which is indicative of the effectiveness of a treatment or treatments.
  • the present invention relates to the use of the kit or the system for the stratification of individuals undergoing drug therapy or being ex- posed to environmental or occupational poisons.
  • the present invention provides the use of the kit or the system for the prediction of safety, toxicity and/or efficacy of a substance, in particular of a drug or prodrug in drug therapy, or of candicate substances for use as active ingredients in pharmaceutical compositions or non- pharmaceutical compositions.
  • the present invention provides a number of advantages.
  • the methods described herein allow for use of a determination an individual's genotype, e.g. of a patient's genotype for the timely administration of the most suitable therapy for that particular individual.
  • the methods of this invention provide a basis for successfully developing and obtaining regulatory approval for a compound even though efficacy or safety of the compound in an unstrati- fied population is not adequate to justify approval.
  • the present invention allows for improving preclinical and clinical development of therapeutics by prospective selection of individuals to be treated.
  • the prospective screening of individuals reduces the risk of unwanted side effects leading to an increased likelihood of successfully developing and registering a compound or composition of compounds.
  • the genetic stratification of individual in advance would allow circumventing difficulties normally occurring during clinical development, such as poor efficacy or toxicity for a subset of the tested cohort.
  • the advantages of a clinical research and drug development program that include the use of polymorphic genotyping for the stratification of individu- als, in particular of patients undergoing a drug therapy, for the appropriate selection of candidate therapeutic intervention includes 1) identification of individuals that may respond earlier to therapy, 2) identification of the primary gene and relevant polymorphic variance that directly affects efficacy, safety, or both, 3) identification of pathophysiologic relevant variance or variances and poten- tial therapies affecting those allelic genotypes, and 4) identification of allelic variances or haplotypes in genes that indirectly affects efficacy, safety or both.
  • determining the presence of the polymorphism(s) accord- ing to the present invention will allow the design of clinical studies being more appropriate to demonstrate the efficacy and safety of the tested compound finally leading to the approval of the compounds or compositions as pharmaceutical products.
  • the method according to the present invention allows for pre- dieting the potential risk of and/or for the diagnosis of carcinomas in the gastrointestinal (colon cancer, pancreatic cancer, hepatocellular cancer, biliary cancer, gastric cancer, esophageal cancer, oropharyngeal cancer) and respiratory (lung cancer) tract and other sites of the human body in addition to chronic inflammatory diseases which include inflammatory bowel disease on the basis said genetic disposition.
  • gastrointestinal colon cancer, pancreatic cancer, hepatocellular cancer, biliary cancer, gastric cancer, esophageal cancer, oropharyngeal cancer
  • respiratory (lung cancer) tract and other sites of the human body in addition to chronic inflammatory diseases which include inflammatory bowel disease on the basis said genetic disposition.
  • Genomic DNA was isolated from full blood samples of patients referred for suspected Gilbert's syndrome by the NucleoSpin Blood XL Kit according to the recommendations of the manufacturer (Machery and Nagel, Dueren, Ger- many). Approximately 40 ng were used as a template in Taqman 5' nuclease assay. Primers and probes specific for (TA) 6 and (TA) 7 , respectively, were designed with the Primer Express software (Applied Biosystems, Darmstadt, Germany) and labeled either 6-FAM or VIC as reporter dyes and with a non- fluorescent quencher (NFQ) and a minor groove binding molecule (MGB) (Ap- plied Biosystems), the respective concentrations were optimized for allelic discrimination and are reported in Table 1.
  • the detection run consisted of a hot start at 95°C for 10 min and 55 cycles of 94°C for 15 s and 60 0 C for 1 min. All assays were performed as 25- ⁇ l reactions using qPCR Mastermix Plus (Euro- gentec, Seraing, Belgium) supplemented with 600 ⁇ M magnesium chloride to a final concentration of 5.6mM of magnesium chloride on 96-well plates using an ABI 7000 instrument (Applied Biosystems).
  • Results were confirmed by direct DNA sequencing after amplification of an 184bp product of the UGT1A1 promoter region followed by sequence PCR.
  • Primer sequences were 5'-TCC CTG CTA CCT TTG TGG AC-3' (forward and sequence PCR) SEQ ID NO. 5 and 5'-AGC AGG CCC AGG ACA AGT-3' (reverse) SEQ ID NO. 6.
  • Sequence PCR was performed using the DyeTerminator Cycle Sequencing Kit 1.1 (Applied Biosystems). The nucleotide sequences were determined on an ABI 310 automated sequencer (Applied Biosystems).
  • UGT1A1*28 represents a challenge for probe design.
  • Factors which complicate the Taqman probe binding and detection include insertional mutations which enable binding of the non-specific probe by causing hairpin structures as well as multiple TA repeats leading to formation of probe homo- and heterodimers, which can inhibit binding to the target sequence. The following adjustments had to be made to the standard protocol in order to ensure an adequate efficiency:
  • TA repeats have a low binding affinity and therefore lead to a reduced binding temperature of short probes.
  • Long Taqman probes are less suitable for allelic discrimination because the mismatching oligonucleotide(s) have to contribute substantially to the binding affinity to allow sufficient discrimination between the differing genotypes.
  • MGB-NFQ Applied Biosystems
  • MGB-NFQ has been used as a quencher in order to gain a higher binding temperature with short probes. Due to different binding kinetics of the wildtype and variant probe, the respective probe concentrations as well as the amount of primers had to be modified, and magnesium added to increase specificity. In addition, the PCR run had to be extended to 55 cycles in order to gain efficient differing fluorescent signals.
  • the resulting cluster plot shows strong fluorescent signals for each allele and a clear separation between the three clusters discriminating the variant, heterozygous and wildtype UGT1A1 * 28 status (figure 1 ).
  • the assay showed low inter-probe variability with a maximum standard deviation of 8.4% (figure 2).
  • Inter-assay variation for the same probes was higher presumably due to naturally occuring hydrolysis of Taqman probes after repeated freeze- thaw-cycles (figure 3). This shows that preferably additional genotyping of positive controls with each run is conducted.
  • the method according to the present invention permits the analysis of up to 44 patients (duplicates including two no- template controls, and two positive controls for wildtype, heterozygous, and homozygous status) in a single run using a standard 96-well-plate. 291 patients with isolated raised bilirubin levels and therefore suspected
  • the signal of the 6-Fam-labled probe specific for the wildtype allele in patients with a heterozygous variant was not high enough to falsely identify them as wildtype.
  • allelic discrimination including poor qual- ity of DNA have to be excluded
  • the method according to the present invention appears suitable for the detection of homozygous as well as heterozygous individuals with the rare UGT1A1 (TA) 5 genotype. In these cases direct sequencing may ensue in case of low dye signals, which would be expected in very few pa- tient samples.
  • the clinical need for genotyping for the (TA) 7 variant of the UGT1A1 promoter region is being increasingly recognized as a predictive factor of standard therapy with certain drugs such as atazanavir and irinotecan.
  • the FDA have already recommended a reduced initial dose of the anti-tumor agent iri- notecan for patients known to be homozygous for the UGT1A1*28 allele (www.fda.gov) (O'Dwyer, P.J. and R. B. Catalano, J. Clin. Oncol. 2006, 24(28): p. 4534-8).

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Abstract

La présente invention concerne selon un premier aspect un procédé permettant de génotyper chez un individu les variantes (TA)7 de la région du promoteur UGT1A1. La présente invention concerne plus particulièrement un procédé spécifique utilisant le système TaqMan d'amplification en chaîne par polymérase de façon à génotyper chez un individu la présence du génotype (TA)6 ou (TA)7 UGT1A1. L'invention concerne également, selon un autre aspect, un nécessaire permettant de génotyper chez un individu des variantes de la région du promoteur UGT1A1, ainsi que des systèmes permettant de les génotyper. Ces nécessaires et systèmes conviennent particulièrement à la stratification d'individus suivant une pharmacothérapie ou professionnellement exposés à un environnement toxique. Selon un autre mode de réalisation, ces procédés, systèmes et nécessaires conviennent à la prédiction de l'innocuité, de la toxicité et/ou de l'efficacité d'une substance, en particulier d'un médicament ou d'un produit utilisés dans une pharmacothérapie, ou de substances destinées à s'utiliser comme principes actifs d'applications pharmaceutiques ou non-pharmaceutiques.
PCT/EP2009/006479 2008-09-05 2009-09-07 Procédé de génotypage de variantes de région du promoteur ugt1a1*28, et systèmes à cet effet WO2010025952A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107043808A (zh) * 2017-01-19 2017-08-15 上海赛安生物医药科技有限公司 Ugt1a1基因多态性检测引物肽核酸及其试剂盒
WO2018235937A1 (fr) * 2017-06-22 2018-12-27 国立大学法人山口大学 Procédé de prévision des effets thérapeutiques de l'irinotécan, et kit destiné à ce procédé

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999057322A2 (fr) * 1998-05-07 1999-11-11 Axys Pharmaceuticals, Inc. Genotypage du gene de l'udp-glucuronosyltransferase 1 humain (ugt1)
US20080153093A1 (en) * 2006-09-19 2008-06-26 Toyo Kohan Co., Ltd. Method for determining risk of adverse effect of irinotecan and kit for it

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999057322A2 (fr) * 1998-05-07 1999-11-11 Axys Pharmaceuticals, Inc. Genotypage du gene de l'udp-glucuronosyltransferase 1 humain (ugt1)
US20080153093A1 (en) * 2006-09-19 2008-06-26 Toyo Kohan Co., Ltd. Method for determining risk of adverse effect of irinotecan and kit for it

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EHMER U ET AL.: "Rapid allelic discrimination by TaqMan PCR for the detection of the Gilbert's syndrome marker UGT1A1*28", JOURNAL OF MOLECULAR DIAGNOSTICS, vol. 10, no. 6, November 2008 (2008-11-01), pages 549 - 552, XP002559014 *
LANKISCH T O ET AL.: "Gilbert's syndrome and irinotecan toxicity: Combination with UDP-Glucuronosyltransferase 1A7 variants increases risk", CANCER EPIDEMIOLOGY BIOMARKERS PREVIEW, vol. 17, no. 3, March 2008 (2008-03-01), pages 695 - 701, XP002559013 *
LANKISCH T O ET AL: "Gilbert's disease and atazanavir: From phenotype to UDP-glucuronosyltransferase haplotype", HEPATOLOGY, WILLIAMS AND WILKINS, BALTIMORE, MD, US, vol. 44, no. 5, 1 November 2006 (2006-11-01), pages 1324 - 1332, XP008096153, ISSN: 0270-9139 *
LANKISCH T O ET AL: "Gilbert's syndrome and hyperbilirubinemia in protease inhibitor therapy - An extended haplotype of genetic variants increases risk in indinavir treatment", JOURNAL OF HEPATOLOGY, MUNKSGAARD INTERNATIONAL PUBLISHERS, COPENHAGEN, DK, vol. 50, no. 5, 1 May 2009 (2009-05-01), pages 1010 - 1018, XP026044597, ISSN: 0168-8278, [retrieved on 20090305] *
LANKISCH T O ET AL: "IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL TATA BOX POLYMORPHISM OF THE UDP GLUCURONOSYLTRANSFERASE 1A7 GENE", MOLECULAR PHARMACOLOGY, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, BALTIMORE, MD, US, vol. 67, no. 5, 1 May 2005 (2005-05-01), pages 1732 - 1739, XP008056864, ISSN: 0026-895X *
VON AHSEN N ET AL.: "DNA base bulge vs unmatched end formation in probe-based diagnostic insertion/deletion genotyping: Genotyping the UGT1A1 (TA)n polymorphism by real-time fluorescence PCR", CLINICAL CHEMISTRY, vol. 46, 2000, pages 1939 - 1945, XP002559012 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107043808A (zh) * 2017-01-19 2017-08-15 上海赛安生物医药科技有限公司 Ugt1a1基因多态性检测引物肽核酸及其试剂盒
WO2018235937A1 (fr) * 2017-06-22 2018-12-27 国立大学法人山口大学 Procédé de prévision des effets thérapeutiques de l'irinotécan, et kit destiné à ce procédé
JP2019004743A (ja) * 2017-06-22 2019-01-17 国立大学法人山口大学 イリノテカンの治療効果予測方法及びそのためのキット
CN110997945A (zh) * 2017-06-22 2020-04-10 国立大学法人山口大学 伊立替康的治疗效果预测方法及应用该方法的试剂盒
EP3643791A4 (fr) * 2017-06-22 2021-03-17 Yamaguchi University Procédé de prévision des effets thérapeutiques de l'irinotécan, et kit destiné à ce procédé
US11384385B2 (en) 2017-06-22 2022-07-12 Yamaguchi University Method for predicting therapeutic effects of irinotecan, and kit for same

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