WO2004018707A2 - Procede pour identifier des polymorphismes d'un seul nucleotide (snp) dans des genes du metabolisme de medicaments, et kit de test pour la mise en oeuvre dudit procede - Google Patents

Procede pour identifier des polymorphismes d'un seul nucleotide (snp) dans des genes du metabolisme de medicaments, et kit de test pour la mise en oeuvre dudit procede Download PDF

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Publication number
WO2004018707A2
WO2004018707A2 PCT/DE2003/002699 DE0302699W WO2004018707A2 WO 2004018707 A2 WO2004018707 A2 WO 2004018707A2 DE 0302699 W DE0302699 W DE 0302699W WO 2004018707 A2 WO2004018707 A2 WO 2004018707A2
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fitc
allele
pcr
specific
labeled
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PCT/DE2003/002699
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German (de)
English (en)
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WO2004018707A3 (fr
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Ralf Neunaber
Pavel Strohner
Joachim Schreiber
Gesine Voigt
Wolf-Hagen Schunck
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Biotez Berlin-Buch Gmbh
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Priority to AU2003269675A priority Critical patent/AU2003269675A1/en
Publication of WO2004018707A2 publication Critical patent/WO2004018707A2/fr
Publication of WO2004018707A3 publication Critical patent/WO2004018707A3/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the subject matter of the invention is a set of substances and prescriptions - overall such a set is also referred to as a "kit” - which allows certain polymorphisms in the cytochrome P450 genes (CYP) of a person which are important for drug metabolism to be reliably detected by simple means.
  • kits which allows certain polymorphisms in the cytochrome P450 genes (CYP) of a person which are important for drug metabolism to be reliably detected by simple means.
  • Areas of application are molecular biology and medicine, especially medical diagnostics.
  • SNPs single nucleotide polymorphisms
  • a large part of the medications used today, especially those with a lipophilic character, are derived from the phase I enzymes of the biotransformation system, the cytochrome P450 Enzymes, metabolized [Gonzales, FJ, Pharmacological Reviews 40, (1989) 243-288; Guengerich et al., Journal of Biological Chemistry 266 (1991) 10019-10022].
  • cytochrome P450 enzymes are divided across families based on their amino acid sequence homology into families (homology> 40% - denoted with an Arabic numeral) and subfamilies (homology> 55% - denoted with a capital letter).
  • CYP2C9 is a family 2, subfamily C, isoform # 9 cytochrome [Nelson et al. Pharmacogenetics 6 (1996) 1-42].
  • the sometimes extremely high sequence homology in genes of a subfamily must also be given priority when developing genotyping methods that are based on amplification of partial gene sequences.
  • the pseudogenes CYP2D7 and CYP2D8 are> 90% identical to CYP2D6. Reliable detection of individual alleles therefore requires reliable differentiation of the homologous genes (isoforms). Technically, this is possible using discriminatory PCR amplification methods.
  • CYP2C9, CYP2C19, CYP2D6 and CYP3A4 with their diverse allele variants are among the best characterized and most important for drug metabolism.
  • Experts estimate that these four isoenzymes together convert more than two thirds of all effective components of today's common drugs.
  • a clear compilation of the implemented pharmaceuticals can be found under [http://medicine.iupui.edu/flockhart/].
  • the alleles in the Caucasian population are CYP2C9 * 2, CYP2C9 * 3, CYP2C19 * 2, CYP2D6 * 3, CYP2D6 * 4, 2D6 * 5, 2D6 * 6 and other, less well-studied alleles of CYP2D6 of highest importance [Wormhoudt et al. Critical Reviews in Toxicolgy 29 (1999) 59-124; Ingelman-Sundberg M. Toxicology Letters 102-103 (1998) 155-160; Marez et al. Pharmacogenetics 7 (1997) 193-202; De Morais et al. Mol. Pharmacol. 46: 594-598 (1994); Sachse et al.
  • allelic variants of CYP3A4 are currently the subject of intensive research [Eiselt et al. Pharmacogenetics 11 (2001) 447-458; Kuehl et al. Nature Genetics 27 (2001) 383-391; Westlind et al. Biochem. Biophys. Res. Cons. 259 (1999) 201-205].
  • Allelic variants of the cytochrome P450 isoenzymes involved in drug metabolism were initially detected with the aid of the sequence comparison of corresponding isolated cDNAs, or by direct sequencing of PCR fragments obtained from genomic DNA [Sullivan-Klose et al. Pharmacogenetics 6 (1996) 341-349; Wang et al. Pharmacogenetics 5 (1995) 37- 42; Inoue et al. Jpn. J. Hum. Genet. 39 (1994) 337-343].
  • a heterologous expression of pre-sequenced or modified cDNA's in suitable systems and subsequent in vitro characterization could then often prove a connection to drug metabolism [Haining et al. Arch. Biochem. Biophys. 333 (1996) 447-458].
  • Another method for identifying new SNPs uses the sequence-dependent mobility of single-stranded PCR products in urea-polyacrylamide gels to detect mutations [Stubbins et al. Pharmacogenetics 6 (1996) 429-439 ; Daly et al. Methods Enzymol. 272 (1996) 199-210].
  • a method which has been used frequently in the past for the detection of SNPs is the so-called "Southern blot".
  • a further development of this principle are the "microarray" gene chip processes which are currently still being developed. Both processes are based on an allele-specific hybridization reaction on immobilized PCR fragments or detection probes. In the case of the "microarray” process, the areas of hybridization are by special occupancy processes sometimes only a few nm in diameter.
  • Another, increasingly used, method for the detection of SNPs makes use of the “matrix-assisted laser desorption / ionization time-of-flight mass spectrometry" (MALDI / TOF-MS).
  • this method also uses an isoform-specific PCR, a subsequent one Purification and allele detection using, for example, hybridized, synthetic oligonucleotides and the subsequent allele-specific incorporation of additional nucleotides by a DNA polymerase beforehand.
  • the sequencing method is comparatively very complex and particularly suitable for the identification of new SNPs. However, it does not detect duplications or deletions of complete genes.
  • the SSCP procedure can capture both and is therefore the method of choice for the early identification of SNPs, but also requires a lot of work or a high degree of automation and thus causes high costs.
  • the RFLP method often requires the replacement of additional nucleotides in the vicinity of the all-terminating position by means of PCR.
  • the digestion is also incomplete in some cases and requires additional validation by means of a positive control or cross-comparison with other alleles.
  • TaqMan technology (Applied Biosystems) - principle: fluorescence detection using a medium-sized hybridization probe.
  • the alley is detected via the 5'-3 'exonuclease activity of a DNA polymerase, which releases quenched 5'-terminal fluorophores (FAM and VIC TM) on perfectly fitting primers. Mismatched primers are released completely intact (i.e. quenched) by the DNA polymerase under the selected conditions, while properly paired primers are released in the unquenched, fluorescent state.
  • FAM and VIC TM quenched 5'-terminal fluorophores
  • the GeneChip CYP450 assay (Affimetrix) is based on a hybridization reaction of allele-specific probes that are immobilized on a “microarray chip” with fluorophore-labeled amplicons.
  • the labeled amplicon is derived from an upstream multiplex PCR, which is intended to ensure isoform-specific amplification via intron-localized primers CYP2C9 * 2 and * 3, as well as 10 alleles of the gene CYP2D6 (from exon 1-9) are offered as complete approaches.
  • the use of this technology requires the very cost-intensive GeneChip Instrument System, as well as the associated GeneChips, reagents - and primer kits.
  • the Code Link TM P450 method (Motorola) is based on the same detection principle as the Affimetrix chip, but uses the detection of 75 CYP450 SNPs (CYP2D6, -2C19, -1A1.1-A2, -2E1, -3A4 and -1B1) a glass slide. It also requires the additional purchase of the very expensive instrumentation and of course the associated reagents and primer kits.
  • the Pharm-O-Kin chip from GenScan Diagnostics is a gene chip based on a glass slide and detects 36 SNP's different P450 isoenzymes as well as other genes relevant for drug transport (CYP2D6, CYP2C9, CYP2C19, as well as NAT2 and MDR1).
  • Orchid's SNP-IT system is a device system that was explicitly developed for the medium to high throughput of SNP detection on PCR amplicons. It essentially consists of a robot, which should automatically process the samples, detect the alleles (a wide variety of technologies such as gene chip hybridization assays, sequencing, etc. can be integrated) and perform data analysis (along with statistics). The acquisition costs for these complex devices are correspondingly high.
  • the SureScore TM system from Invitrogen is a SNP detection system based on 96 well plates.
  • the basic technology is the specific incorporation of labeled (biotin or FITC) dideoxynucleotides on a single-stranded, complementary PCR product matrix by a DNA polymerase at the SNP position.
  • the subsequent detection is carried out by a marker-specific antibody that catalyzes a color reaction via a coupled enzyme.
  • the invention is therefore based on the object of developing such a method and at the same time making a test kit available for carrying out the method
  • the invention is implemented according to the claims. It concerns a simplified procedure for the detection of SNP in human CYP genes and a test kit with the following components:
  • Newly developed synthetic oligonucleotides preferably oligonucleotides from Appendix 1, which are suitable for amplifying gene sections in genes of drug metabolism with the aid of the “DNA polymerase chain reaction” technology (PCR) isoform-specifically (IS-PCR) from genomic leukocyte DNA,
  • PCR DNA polymerase chain reaction
  • IS-PCR isoform-specifically
  • FITC fluorescein isothiocyanate
  • SA-MTP's from streptavidin-coated glass slides (SA chip) on which biotinated, isoform-specific, synthetic oligonucleotides are immobilized, which are hybridized with partially single-stranded PCR products which are discriminated by the extension of an allele-specific, terminally FITC-labeled oligonucleotide using Taq DNA polymerase were generated and, by subsequent stringent washing and subsequent detection using fluorometry or photometry, ensure reliable genotyping of the hybridized amplicons.
  • the invention described here makes it possible to collect the therapeutically important findings for individual equipping with the various CYP alleles, even in a few individual patients with low personnel and material costs.
  • the invention uses highly gene-specific oligonucleotide primers, preferably oligonucleotides from Appendix 1, for flanking regions of the allele-determining gene segments in order to amplify them from a background of very homologous cytochromes of the same subfamily by means of discriminatory PCR.
  • the following allele detection can be done using two different methods:
  • biotinated gene-specific oligonucleotides are immobilized on defined spots of a glass slide coated with streptavidin, and the partially single-stranded duplexes are subsequently immobilized with the gene-specific Oligonucleotides hybridized and freed from stringent washing of non-extended, 5'-FITC labeled oligonucleotide primers.
  • the primers and probes used according to the invention enable highly selective allele detection of genomic DNA within a few hours
  • test is carried out as a multiple assay, in which a plurality of individual alleles are detected simultaneously in a specially adapted protocol run on an MTP or several glass object carriers, and
  • Samples consisting of genetically engineered plasmid DNA (pDNA), preferably the plasmid DNA sequences of Appendix 1, which contain allele-specific fragments of CYP gene segments, can be carried as internal controls.
  • pDNA genetically engineered plasmid DNA
  • Appendix 1 plasmid DNA sequences of Appendix 1, which contain allele-specific fragments of CYP gene segments
  • Genomic DNA can be isolated from leukocytes of human blood using standard methods already described (Vogelstein B. and Gillespie D. PNAS, (1979) 76: 615-9).
  • a gDNA concentration of 5-50 ng / ul should be aimed for in order to present a sufficient number of copies when using 0.5-1 ⁇ l gDNA / PCR.
  • the quality of the gDNA can be of crucial importance for the effectiveness of the entire assay and therefore the quality and concentration should be as constant as possible.
  • a 10-fold concentrated polymerase buffer e.g. 100 mM Tris-HCl (pH 8.0), 500 mM KCI, 15 mM MgCI); 0.08 ⁇ l dNTP (62.5 mM each); 0.5 ⁇ l each of the 50 ⁇ M primer SP2C9201 (SEQ ID NO 1) and the 5 ′ biotinated primer RP2C9201 (SEQ ID NO 2); 0.5 ul Taq DNA polymerase (5U / ul), 1 ul gDNA; and 16.34 ⁇ l bidest suitable for PCR in a double batch pipetted into PCR tubes.
  • the tubes are then transferred to a commercially available PCR device and subjected to the following PCR regime:
  • samples consisting of genetically engineered plasmid DNA which contain allele-specific fragments of the gene segment CYP2C9 * 2 (SEQ ID NO 38) and the corresponding segment of the wild-type gene CYP2C9 * 1 (SEQ ID NO 37), were also used as internal controls in duplicate carried.
  • the resulting amplification products can be separated by agarose gel electrophoresis in 3.5% agarose gels at 5-10 V / cm gel width for 1-1 1/2 hours and visualized and documented using DNA markers and ethidium bromide staining under UV light.
  • TE-NaCI 10 mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl
  • SDS-WP 0.1 x SSC; 0.1% SDS
  • TE-lysine 10mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl; 2% lysine (w / v)
  • TE-RSA 10mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl; 0.05%
  • Bovine serum albumin (w / v) TE-Tween: 10 mM Tris-HCl (pH 7.5); 1M EDTA; 100 mM NaCl; 0.1% tween
  • hybridization probes final concentration - 0.5 - or 1 nM prediluted in HP (hybridization buffer) are added to each of the double batches, hybridized for 20 minutes at 37 ° C. and then aspirated.
  • the pDNA contains all-specific fragments of the gene section CYP2C19 * 3 (SEQ ID NO 44) or the corresponding section of the wild-type gene CYP2C19 * 1 (SEQ ID NO 43).
  • a 10-fold concentrated polymerase buffer e.g.
  • TE-NaCI 10 mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl
  • SDS-WP 0.1 x SSC; 0.1% SDS
  • TE-lysine 10mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl; 2% lysine (w / v)
  • TE-RSA 10mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl; 0.05%
  • Bovine serum albumin (w / v) TE-Tween: 10 mM Tris-HCl (pH 7.5); 1mM EDTA; 100 mM NaCl; 0.1% tween
  • a SA chip equipped with 32-64 reaction areas (spots) is provided with 3 ⁇ l / spot of a 5 'biotinated 500 pM oligos dissolved in TE-NaCI with the designation B2C193_F (SEQ ID NO 58), 15 minutes at RT incubated in a humidity chamber, then aspirated, washed once with 6 ⁇ SSC at RT.
  • 0.25 ⁇ l of the amplification products obtained in Example 3 are mixed with 1.5 ⁇ l 12 ⁇ SSC, made up to 3 ⁇ l total volume with ddH 2 0, after aspirating the 6 ⁇ SSC buffer in triplicate, pipetted onto individual spots, 30 minutes at RT hybridized with saturated humidity and then aspirated.
  • CYP2C19 * 1 is genetically engineered, double-stranded plasmid DNA with the contained sequence for CYP2C19 WT (SEQ ID NO 43) and accordingly for CYP2C19 * 3 (SEQ ID NO 44);
  • FITC probe 5 'fluoroescein labeled oligonucleotide;
  • Match / Mismatch (Mw) quotient of the average score from 100% complementary and non-complementary allele probe hybrid;
  • nx PE number (s) of FITC primer extension cycles performed.

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Abstract

L'invention concerne un procédé simplifié, servant à l'identification de SNP dans des gènes de cytochromes P450 (CYP) chez l'homme et un kit de test comportant les éléments suivants : 1) de nouveaux oligonucléotides de synthèse, appropriés pour amplifier des segments de gène dans des gènes du métabolisme de médicaments, par PCR (réaction en chaîne de l'ADN polymérase) spécifique des isoformes (IS--PCR) à partir d'ADN génomique de leucocytes ; 2) des éléments qui permettent d'immobiliser des produits IS-PCR biotinylés sous forme de simple brin sur des plaques de microtitration revêtues de streptavidine (SA-MTP) de manière sélective, thermostable et sous forme de simple brin ; 3) des oligonucléotides de synthèse, optimisés pour le test, spécifique de l'allèle considéré, marqués par un isothiocyanate de fluorescéine (FITC), qui assurent un génotypage fiable des produits d'amplification immobilisés (amplicons), par hybridation, suivie d'un lavage stringent et d'une détection par fluorométrie ou photométrie ; ou en variante 4) des porte-objets en verre revêtus de streptavidine (puce SA) sur lesquels sont immobilisés des oligonucléotides synthétiques, spécifiques des isoformes, biotinylés, hybridés par des produits de PCR partiellement simple brin, qui ont été produits par allongement discriminatoire d'un oligonucléotide marqués par un FITC, à l'extrémité terminale, spécifique à l'allèle, au moyen d'une Taq ADN polymérase et qui assurent un génotypage fiable de l'amplicon hybridé, par lavage stringent suivi d'une détection par fluorométrie ou photométrie. A la différence des procédés à haut débit connus, le procédé selon l'invention permet d'obtenir, avec de faibles coûts en personnel et en matériel, même à partir d'un faible nombre de patients individuels, des résultats importants sur le plan thérapeutique, le traitement étant adapté à chaque patient en fonction des différents allèles de CYP.
PCT/DE2003/002699 2002-08-15 2003-08-07 Procede pour identifier des polymorphismes d'un seul nucleotide (snp) dans des genes du metabolisme de medicaments, et kit de test pour la mise en oeuvre dudit procede WO2004018707A2 (fr)

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AU2003269675A AU2003269675A1 (en) 2002-08-15 2003-08-07 Method for identifying single nucleotide polymorphisms (snp) in genes which metabolize medicaments and test kit for carrying out said method

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DE2002137691 DE10237691B4 (de) 2002-08-15 2002-08-15 Verfahren zum Nachweis von Einzelnukleotid-Polymorphismen (SNP) in Genen des Arzneimittelmetabolismus und Testkit zur Durchführung des Verfahrens
DE10237691.3 2002-08-15

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
EP1781812A1 (fr) * 2004-06-30 2007-05-09 TM Bioscience PGX, Inc. Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9

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Publication number Priority date Publication date Assignee Title
AU2005266805B2 (en) * 2004-07-30 2010-10-28 Luminex Molecular Diagnostics, Inc. Method of detecting mutations in the gene encoding Cytochrome P450-2C19

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
EP1781812A1 (fr) * 2004-06-30 2007-05-09 TM Bioscience PGX, Inc. Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9
EP1781812A4 (fr) * 2004-06-30 2008-10-15 Tm Bioscience Pgx Inc Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9
AU2005259786B2 (en) * 2004-06-30 2011-03-10 Luminex Molecular Diagnostics, Inc. Method of detecting mutations in the gene encoding cytochrome P450-2C9

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AU2003269675A1 (en) 2004-03-11

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