EP1230386A2 - Kit de diagnostic, procede et jeu ordonne d'echantillons permettant de determiner l'aptitude humaine a la desintoxication - Google Patents

Kit de diagnostic, procede et jeu ordonne d'echantillons permettant de determiner l'aptitude humaine a la desintoxication

Info

Publication number
EP1230386A2
EP1230386A2 EP00969543A EP00969543A EP1230386A2 EP 1230386 A2 EP1230386 A2 EP 1230386A2 EP 00969543 A EP00969543 A EP 00969543A EP 00969543 A EP00969543 A EP 00969543A EP 1230386 A2 EP1230386 A2 EP 1230386A2
Authority
EP
European Patent Office
Prior art keywords
oligonucleotides
dna
diagnostic kit
enzyme
kit according
Prior art date
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.)
Withdrawn
Application number
EP00969543A
Other languages
German (de)
English (en)
Inventor
Stefanie WASCHÜTZA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alere Diagnostics GmbH
Original Assignee
Adnagen GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Adnagen GmbH filed Critical Adnagen GmbH
Publication of EP1230386A2 publication Critical patent/EP1230386A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—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
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a diagnostic kit, a method, a microarray and their
  • Diagnostic kits, methods and microarrays of this type are used in the field of environmental medicine and occupational medicine and the associated diseases such as neurodermatitis, asthma, MCS (multiple chemical sensitivity, multiple chemical sensitivity) or CFS (chronic fatigue syndrome, fatigue syndrome). They are also used in the field of pharmacology or to determine the human metabolism of medication or new products.
  • phase I reaction the order ⁇ world poisons initially by enzymes from the Zytochromsy- stem m reactive intermediates implemented, but which can be acutely toxic than the contaminants themselves.
  • These intermediates of the phase I reaction can, for example, to cellular components bind and thereby cause damage.
  • chronic damage is usually due to the water-insoluble starting substances.
  • phase II subsequent to phase I
  • the reaction converts the reactive intermediate products into end products that can be separated.
  • Phase I describes the implementation and activation of various known drugs and environmental chemicals (e.g. debrisoqum, opioids, Benzo (a) pyrene, polycyclic and aromatic hydrocarbons) and a number of as yet unknown xenobiotics to electrophilic, radical substances.
  • drugs and environmental chemicals e.g. debrisoqum, opioids, Benzo (a) pyrene, polycyclic and aromatic hydrocarbons
  • phase II reactions There are several reaction pathways in phase II reactions, including glucoronidation, sulfate ester formation, amino acid conjugation, the enzymes of the most important phase II reaction pathway (glutathione S transferases) being the intermediates from phase I (e.g. Deactivate epoxides, nitrosams) to end products that can be eliminated by conjugation with glutathione.
  • glutathione S transferases the enzymes of the most important phase II reaction pathway (glutathione S transferases) being the intermediates from phase I (e.g. Deactivate epoxides, nitrosams) to end products that can be eliminated by conjugation with glutathione.
  • glutathione-S-transferase This deactivates intermediates from the phase I reaction to end products which can be excreted by conjugation with glutathione, the reduced form of which has a nucleophilic SH group which easily reacts with electrophilic carbon atoms to form the thioether.
  • the highest possible activity of glutathione S-Transf erase means efficient conjugation and rapid detoxification, while a reduced or no activity of the Glutathione-S-Transferase causes a slow detoxification.
  • the system of glutathione-S-transferase has several different enzymes, for example the glutathione-S-transferase GSTM1 and the glutathione-S-transferase GSTT1.
  • the glutathione-S-transferase GSTM1 has a polymorphism in which the individual genotypes coexist on an equal footing.
  • Genotype 00 indicates the lack of the gene for GSTM1. If there is a functional gene, two different variants A and B can be distinguished. Both variants form active homodimeric enzymes (AA / BB or in the absence of the gene for an allele A0 / B0) or heterodimeric enzymes (AB), the mixed form AB showing the highest enzyme activity and, among other things, is made responsible for the "drug-resistance" phenomena in chemotherapy.
  • Glutathione-S-Transferase Ml plays a major role in the liver, lungs and skin, with the null genotype occurring in about 40% of the Caucasian population. This zero genotype is described with the development of various tumor diseases (lung, skin, and bladder cancer) as well as chronic diseases with environmental pollution factors (asbestosis, chronic bronchitis).
  • Glutathione-S-Transferase GSTT1 The glutathione-S-Transf erase Tl comes u. a. in the liver and erythrocytes and mediates glutathione conjugation with halogenated hydrocarbons and epoxides. No enzyme activity of glutathione-S-transferase was found in erythrocytes in about 25% of the German population examined to date. Genotype 00 indicates the lack of the gene for this glutathione S-transferase. The O genotype is therefore regarded as a risk factor because the GSTT1-mediated activity against DNA-damaging peroxides and other metabolites is reduced.
  • Known toxic substrates of GSTT1 include a. Methyl bromide, ethylene oxide and methylene chlorine id.
  • a stress test is carried out according to the state of the art, which can be used to determine the detoxification ability and thus enzyme activity of this individual.
  • this is done by administering a test substance that is broken down by the enzymes to be examined and then determining the clearance of the individual in question for this substance.
  • the disadvantage of this is that the determination of the enzyme activities can only be determined by additional exposure of the person to a chemical. Such a method of determination is particularly forbidden for children or people with a reduced general condition or people who are known to be heavily polluted. Therefore, this method cannot be used in every case, especially not in persons for whom the determination of the enzyme activity would be particularly important for determining the appropriate therapy due to their pollution.
  • DE 197 38 908 which goes back to the same inventors as the present invention, proposes to determine genetic polymorphisms of genes in individuals which code for an enzyme from the detoxification metabolism.
  • the polymorphism can consist of both the creation of a gene and m different allelic variabilities of one or more polymorphic genes.
  • the object of the present invention is therefore to provide a diagnostic kit, a microarray, a method and uses thereof, with which the human detoxification ability can be determined in a simple manner by any laboratory in a standardized manner.
  • the diagnostic kit according to the invention contains the substances required to carry out a polymerase chain reaction, as are also offered by various manufacturers. Furthermore, the diagnostic kit contains at least one pair of oligonucleotides, which are required as a reversepimer and forward primer for the amplification by means of the polymerase chain reaction. Such oligonucleotides are also used in the methods according to the invention. The oligonucleotides of the pairs are selected according to the invention in such a way that an amplification of a DNA segment takes place which codes for at least part of the enzyme GSTT1 of the animal detoxification metabolism. If an DNA segment is thus amplified, it can simply be determined whether a gene for the enzyme GSTT1 is present. This enables a zero-type determination.
  • oligonucleotides are used in the methods according to the invention or are given in the diagnostic kit for each of the alleles, so that an amplification of an allele can be detected. This can also be done, for example, in such a way that the oligonucleotides for the individual alleles differ in such a way that regions of the DNA of the alleles are amplified which differ, for example, by an interface for a restriction enzyme.
  • the detection of a gene or its alleles can also be carried out using the microarray according to the invention, e.g. of a DNA chip, with the individual cells of the chip having oligonucleotides that hybridize specifically to certain sections of the genes to be detected.
  • the detection can take place directly without amplification or only after amplification of the DNA section sought. In the same way, the detection can be carried out without or only after restriction digestion of the DNA or an amplified DNA section thereof into shorter DNA sections.
  • An advantage of the diagnostic kit according to the invention, the method according to the invention and the microarray according to the invention is that every laboratory doctor and every medical laboratory as well as every scientific laboratory which carries out such tests is provided with all coordinated substances, if necessary in a single diagnostic kit , or that any preparatory work for the laboratories is omitted and simple and
  • the polymorphism can be determined.
  • the polymer oligonucleotides or the oligonucleotides of the microarray have already been tested, so that the main work in developing corresponding oligonucleotides is omitted.
  • thermoscycler PCR device
  • a device for separating the individual amplified DNA fragments can be, for example, a gel electrophoresis unit or a capillary electrophoresis device.
  • a conventional capillary electrophoresis device is marketed, for example, by the company Per in Eimer Biosystems TM under the name "PE ABI P ⁇ sm Genetic Analyzer 310" TM.
  • Corresponding thermocyclers for amplifying the DNA are sold, for example, by the company Perkin Elmer Biosystems TM under the name "GenAmp 2400" TM or "GenAmp9600" TM.
  • At least one of the oligonucleotides of a pair can be labeled with a fluorophore, so that an automated evaluation based on the specific fluorescence emission of the respective fluorophores is also possible.
  • diagnosis kit, microarray and method according to the invention enable everyone to determine the polymorphism of detoxification enzymes in a simple, safe and standardized manner. not.
  • the diagnostic kit can also be developed in such a way that not only oligonucleotides for the determination of a single enzyme but also other oligonucleotide pairs for the determination of further genes of detoxification enzymes are added to the diagnosis kit.
  • the microarray according to the invention can also provide fields which have oligonucleotides with which further genes of detoxification enzymes can be detected.
  • kits and methods according to the invention are to be described below.
  • a first kit enables a multiplex zero type determination by means of PCR (polymerase chain reaction) of the genes for GSTM1 and GSTT1 with labeled oligonucleotides as primers for detection via the device Perkin Elmer ABI Prism Genetic Analyzer 310.
  • the kit contains the following ingredients separately in individual containers: 10 ⁇ l PCR buffer (10 x buffer, e.g. from Promega)
  • 2 ⁇ l dNTPs (10 mM) (deoxynucleotide triphosphates, e.g. guanine (G), adenine (A),
  • Ta ⁇ r polymerase 5U / ⁇ l (e.g. from Promega)
  • the oligonucleotides GSTMl fw which serve as forward primers, are labeled with the dye fern (carboxyfluorescein), the forward primer for albumin is labeled with Ned (fluorescent dye from PE Bio-systems) and the forward Primer for GSTT1 marked with hex (4, 7, 2 ', 4', 5 ', 7' -hexachloro-6-carboxy-fluorescein).
  • the individual primers have the following nucleotide sequences:
  • GSTMl fw GAA CTC CCT GAA AAG CTA AAG C;
  • GSTMl rv GTT GGG CTC AAA TAT ACG GTG G;
  • GSTT1 fw TTC CTT ACT GGT CCT CAC ATC TC;
  • GSTT1 rv TCA CCG GAT CAT GGC CAG CA;
  • the kit also contains the description of the procedure for amplifying the DNA with the following parameters:
  • the evaluation can now be carried out, for example, using gel electrophoresis.
  • a 2.5% gel (SeaKem LE TM, Biozym TM) is poured, a marker (100 bp ladder) is also applied and the gel is run at a voltage of 100 mV for 45 minutes for separation.
  • the internal control for the gene for albumin is 350 bp in length and must be positive in every approach.
  • the GSTML has a length of 219 bp and the GSTT1 has a length of 480 bp.
  • the last two genes can only be seen as DNA bands in the gel if a functional gene is present, otherwise the respective null type is present.
  • Lane 1 shows such a gel, in which the bands for the GSTT1 (Tl) at 480 bp, for the gene for albumin (A) at 350 bp and for the gene GSTMl (Ml) at 219 bp clearly in lanes 3 and 4 are recognizable.
  • Lane 2 contains standard marker substances. In this case, therefore, both the gene for GSTMl and the GSTTl gene are present.
  • FIG. 2 shows a corresponding evaluation by means of capillary electrophoresis using the PE ABI Prism Genetic Analyzer 310 TM.
  • the corresponding bands can be clearly seen at the corresponding fragment lengths (in base pairs).
  • the heights of the individual bands depend on the fluorescent markers used and therefore do not provide any quantitative information.
  • the other bands represent the ROX standard, which enables the fragment sizes to be assigned.
  • FIG. 3 shows the results of a further investigation using capillary electrophoresis.
  • the bands of the Rox standard only the bands of the albumin standard (A) and the GSTT1 (Tl) occur. Obviously there is a null type with regard to the GSTM1.
  • a kit was used for the variant determination of the GSTML with labeled oligonucleotides as primers.
  • Glutathione-S-Transferase Ml is characterized by a genetic polymorphism in two ways.
  • one or both of the genes GSTM1 coding for the glutathione-S-transferase Ml may be missing and thus one type homozygous if two genes GSTMl are present, one heterozygous type with only one gene GSTMl and one in the absence of both Genes in turn are homozygous null type.
  • the exchange of guanine (G) for cytosine (C) at position 2619 results from an enzyme type B to an enzyme type A, the heterozygous AB type having the highest potential enzyme activity.
  • the kit used to determine this allele variability contains the following substances required for PCR, each in separate individual packaging:
  • A Adenine (A), thymine (T)
  • 0.5 ul tag polymerase (5U / ul) (e.g. from Promega)
  • the oligonucleotides GSTM1-A are marked with the fluorophore hex and the oligonucleotides GSTM1-B are marked with the fluorophore fern.
  • the oligonucleotides have the following base sequence:
  • GSTMl fw GCT TCA CGT GTT ATG GAG GTT C for both AI lele the GSTMl;
  • GSTM1-A TTG GGA AGG CGT CCA AGC GC
  • GSTMl -B TTG GGA AGG CGT CCA AGC AG.
  • the kit also contains instructions with the following PCR program:
  • the DNA segments amplified by the above-mentioned PCR program have a length of 132 bp.
  • the detection is again carried out by capillary electrophoresis with the results shown in FIGS. 4 and 5.
  • the band that is formed by the 132 bp DNA fragment of variant B of the GSTM1 and is marked with the fluorophore FAM is identified by Ml-B, while Ml -A denotes the band that by the 132 bp l DNA pieces of variant A of the GSTMl are created.
  • the evaluation can also be carried out via gel electrophoresis.
  • a 2.5 mg gel (Se a-Kern LE Biozym TM) is poured, in which case a marker (100 bp ladder) is then also applied in addition to the PCR product in order to check the PCR success.
  • the gel then runs at a voltage of 100 mV for 45 minutes.
  • the PCR product is subjected to a restriction digest. Because Variant A contains an interface for Haell and is split into two fragments of 116 bp and 16 bp in length, while variant B contains no interface for the restriction enzyme Haell.
  • FIG. 6 shows such a gel for different samples of different gel traces, in which both the 116 bp band ("GSTM1-A" for variant A) and the 132 bp band ("GSTM1-B" for variant B) can be seen .
  • the second lane from the left contains standard marker substances.
  • Lanes 4, 6, 9 correspond to samples containing the gene for GSTM1 variant B, while lanes 3, 5, 7, 8, 10 to 14 contain samples with the gene for GSTMl variant A.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un kit de diagnostic, un jeu ordonné d'échantillons et un procédé permettant de déterminer l'aptitude humaine à la désintoxication. Le kit de diagnostic contient au moins une paire d'oligonucléotides (amorce inverse, amorce en avant), les deux oligo-nucléotides d'une paire convenant comme amorce aux fins d'amplification par une réaction en chaîne de la polymérase respectivement d'un des deux brins complémentaires d'un segment DNS recherché et le segment DNS recherché codant au moins une partie d'un enzyme du métabolisme de désintoxication animal.
EP00969543A 1999-11-16 2000-10-25 Kit de diagnostic, procede et jeu ordonne d'echantillons permettant de determiner l'aptitude humaine a la desintoxication Withdrawn EP1230386A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19955024A DE19955024C2 (de) 1999-11-16 1999-11-16 Diagnose-Kit
DE19955024 1999-11-16
PCT/EP2000/010478 WO2001036670A2 (fr) 1999-11-16 2000-10-25 Kit de diagnostic, procede et jeu ordonne d'echantillons permettant de determiner l'aptitude humaine a la desintoxication

Publications (1)

Publication Number Publication Date
EP1230386A2 true EP1230386A2 (fr) 2002-08-14

Family

ID=7929173

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00969543A Withdrawn EP1230386A2 (fr) 1999-11-16 2000-10-25 Kit de diagnostic, procede et jeu ordonne d'echantillons permettant de determiner l'aptitude humaine a la desintoxication

Country Status (6)

Country Link
EP (1) EP1230386A2 (fr)
JP (1) JP2003517147A (fr)
AU (1) AU7922300A (fr)
CA (1) CA2388563A1 (fr)
DE (1) DE19955024C2 (fr)
WO (1) WO2001036670A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10219373A1 (de) * 2002-04-30 2004-02-19 Adnagen Ag Verfahren und Diagnosekit zur molekularen Diagnostik pharmakologisch relevanter Gene
DE10246339A1 (de) * 2002-10-04 2004-04-15 Adnagen Ag Verfahren zur Vorhersage der Wahrscheinlichkeit an einer Chemikalien-induzierten Krankheit zu erkranken
EP3385393A1 (fr) * 2017-04-05 2018-10-10 Eckart Schnakenberg Procédé in vitro destiné au diagnostic du risque de formation d'un syndrome aérotoxique chez un sujet et kit d'exécution du procédé
CN107475397A (zh) * 2017-09-06 2017-12-15 江苏省疾病预防控制中心 一种检测易感职业性噪声聋基因的方法及其试剂盒

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19738908B4 (de) * 1997-09-05 2004-01-29 Adnagen Gmbh Verfahren zur Bestimmung individueller genetischer Einflüsse für Schadstoffeinwirkungen und/oder Planung eines Therapiekonzeptes für neurodermitis- und/oder asthmagefährdete oder -erkrankte Personen
US5919627A (en) * 1997-10-29 1999-07-06 Incyte Pharmaceuticals, Inc. Microsomal glutathione-S-transferase
WO1999064626A2 (fr) * 1998-06-06 1999-12-16 Genostic Pharma Limited Sondes permettant de determiner un profil genetique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0136670A2 *

Also Published As

Publication number Publication date
DE19955024A1 (de) 2001-06-13
WO2001036670A2 (fr) 2001-05-25
JP2003517147A (ja) 2003-05-20
WO2001036670A3 (fr) 2001-12-06
DE19955024C2 (de) 2003-01-16
CA2388563A1 (fr) 2001-05-25
AU7922300A (en) 2001-05-30

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