EP1358351A2 - Diagnostic de parametres genetiques importants a l'interieur du cmh - Google Patents

Diagnostic de parametres genetiques importants a l'interieur du cmh

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Publication number
EP1358351A2
EP1358351A2 EP01955494A EP01955494A EP1358351A2 EP 1358351 A2 EP1358351 A2 EP 1358351A2 EP 01955494 A EP01955494 A EP 01955494A EP 01955494 A EP01955494 A EP 01955494A EP 1358351 A2 EP1358351 A2 EP 1358351A2
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European Patent Office
Prior art keywords
oligonucleotides
mhc
nucleic acids
diagnosis
pna oligomers
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EP01955494A
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German (de)
English (en)
Inventor
Alexander Olek
Christian Piepenbrock
Kurt Berlin
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Epigenomics AG
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Epigenomics AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to nucleic acids, oligonucleotides, PNA oligomers and a method for the diagnosis of important genetic parameters within the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • MHC Major Histocompatibility Complex
  • Chromosome 6 (6p21.3) and is significantly involved in the immune response. It is completely sequenced, highly polymorphic and has the highest gene density in the entire human genome. Of the total of 3500 genes estimated on chromosome 6, 224 are identified as loci of the MHC, which means that 3 times as many genes ⁇ OM IV) MM cn o Cn o cn O cn
  • HIV-1 Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.
  • MHC major histocompatibility complex
  • CTLA-4 gene polymorphisms is associated with predisposition to coeliac disease. 1998, Djilali-Saiah I et al., Gut; 43 (2): 187-189), Myasthenia gravis, Spondylarthropathia (Genes in the spondyloarthropathies. 1998, Wordsworth P., Rheum Dis Clin North Am; 24 (4): 845-863 .
  • Tuberculosis Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors. 1998, Ulrichs T et al., Eur J Immunol; 28 (12): 3949-3958), hypertrophic cardiomyopathy (Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. 1997, Kimura A., Nat. Genet. 16 (4): 379-382), Graves' disease (Iodi de, cytokines and TSH-receptor expression in Graves' disease. 1996, Schuppert et al. , Exp Clin Endocrinol Diabetes.
  • HLA-DR antigens and the methylation of the HLA-DR alpha gene was investigated in systemic lupus erythematosus, a generalized autoimmune disease (low expression of human histocompatibility leukocyte antigen-DR is associated with hyper-methylation of human histocompatibility leukocyte antigen-DR alpha gene regions in B cells from patients with systemic lupus erythematosus. 1985; Sano H et al., J Clin Invest, 76 (4) .1314-1322).
  • MHC class II deficiency syndrome The involvement of DNA methylation in the aberrant MHC class II gene expression was investigated in patients with MHC class II deficiency syndromes (The MHC class II deficiency syndrome: heterogeneity at the level of the response to 5-azadeoxycytidine. 1990; Lambert M et al., Res Immunol. 141 (2): 129-140).
  • Evidence for the regulation of MHC genes was provided using an epigenetic mechanism (Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control. 2000, Morris AC et. Al., J Immunol; 16 (8 ): 143-4149).
  • the expression of MHC genes is inhibited if transcription factors do not bind to the class 2 trans activator (CIITA) promoter.
  • CIITA class 2 trans activator
  • 5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. Identification of 5-methylcytosine as an ingredient genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing because 5-methylcytosine has the same base pairing behavior as cytosine. In addition, a PCR
  • Methylcytosine is based on the specific reaction of bisulfite with cytosine, which after subsequent alkaline hydrolysis is converted into uracil, which corresponds in its base pairing behavior to thymidine. 5-Methylcytosine, however, is not modified under these conditions. The original DNA is thus converted in such a way that methylcytosine, which originally cannot be distinguished from the cytosine by its hybridization behavior, can now be detected by "normal" molecular biological techniques as the only remaining cytosine, for example by amplification and hybridization or sequencing.
  • Mass spectrometry is a very powerful CO CO IV) IV) MM
  • Genomic DNA is obtained by standard methods from DNA from cell, tissue or other test samples. This standard methodology can be found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
  • the object of the present invention is to provide oligonucleotides and / or PNA oligomers for the detection of cytosine methylations and a method which is particularly suitable for the diagnosis of genetic and epigenetic parameters within the MHC.
  • nucleic acids for the diagnosis of a set of genetic parameters within the major histocompatibility complex comprising a reverse complementary or identical section of the chemically pretreated genomic DNA according to SEQ ID-NO: 1 or SEQ ID-N0 that is at least 20 base pairs long : 2nd
  • the present invention relates to oligonucleotides for the detection of the cytosine methylation state in pretreated genomic DNA, each comprising at least one base sequence with a length of at least 13 nucleotides which are reverse complementary or identical to a section of the base sequences according to SEQ ID-NO: 1 or SEQ ID-NO: 2, which contains at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide from the 5 'end of the 13 mer.
  • an oligonucleotide is present for each of the CpG dinucleotides from one of SEQ ID-NO: 1 or SEQ ID-NO.-2.
  • the present invention relates to PNA (peptide nucleic acid) oligomers for the detection of the cytosine methylation state in chemically pretreated genomic DNA, comprising at least one PNA base sequence with a length of at least 9 nucleotides which are reverse complementary or identical to a section of the base sequences according to SEQ ID-NO: 1 or SEQ ID-NO: 2, which contains at least one CpG dinucleotide.
  • PNA peptide nucleic acid
  • the cytosine of the CpG dinucleotide is the 4th - 6th nucleotide from the 5 'end of the 9 mer.
  • an oligonucleotide is present for each of the CpG dinucleotides from a base sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention relates to a set of oligomer probes for detecting the cytosine methylation state and / or of single nucleotide polymorphisms in chemically pretreated genomic DNA, comprising at least 10 of the oligonucleotide or PNA sequences according to the invention.
  • the present invention also relates to an arrangement of different oligo- according to the invention OO to IV) MM no cn o cn O Cn
  • the chemical treatment is carried out using a solution of a bisulfite, bisulfite or disulfite.
  • the polymerase is a heat-resistant DNA polymerase.
  • the amplification is carried out by means of the polymerase chain reaction (PCR). It is also preferred that the labels attached to the amplificates can be identified at any position of the solid phase at which an oligonucleotide sequence is located. Furthermore, it is particularly preferred according to the invention that an arrangement according to the invention is used and that the solid phase surface consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.
  • the amplification of several DNA sections is carried out in one reaction vessel.
  • the labels of the amplified products are fluorescent labels.
  • the labels of the amplificates are radionuclides.
  • the labels of the amplified products are detachable molecular fragments with a typical mass, which are detected in a mass spectrometer. It is particularly preferred according to the invention that the amplified products or fragments of the amplified products are detected in the mass spectrometer. For this purpose, it is advantageous according to the invention that for better detectability in the mass spectrometer generated fragments have a single positive or negative net charge.
  • the detection is carried out and visualized by means of matrix assisted laser desorption / ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (.ESI).
  • MALDI matrix assisted laser desorption / ionization mass spectrometry
  • .ESI electrospray mass spectrometry
  • the method is preferred, wherein the genomic DNA was obtained from a DNA sample, sources of DNA e.g. B. cell lines, biopsies, blood, sputum, stool, urine, brain spinal fluid, tissue embedded in paraffin, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and includes all possible combinations thereof.
  • sources of DNA e.g. B. cell lines, biopsies, blood, sputum, stool, urine, brain spinal fluid, tissue embedded in paraffin, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and includes all possible combinations thereof.
  • a method for diagnosing and / or predicting adverse events for patients or individuals is also preferred, these adverse events being associated with methylation patterns within the MHC.
  • the present invention also relates to the use of a method according to the invention, wherein important genetic parameters are diagnosed within the MHC.
  • Another object of the present invention is a kit consisting of a. Reagent containing bisulfite, sets of primers according to the invention for producing the amplificates according to the invention, oligonucleotides and / or PNA oligomers and instructions for carrying out and evaluating a method according to the invention.
  • a. Reagent containing bisulfite, sets of primers according to the invention for producing the amplificates according to the invention, oligonucleotides and / or PNA oligomers and instructions for carrying out and evaluating a method according to the invention.
  • the oligonucleotides or PNA oligomers are bound to a solid phase at defined locations.
  • Different amplificates are preferably arranged on the flat solid phase in the form of a right-angled or hexagonal lattice.
  • the nucleic acids, oligonucleotides or PNA oligomers are preferably used to diagnose rheumatoid arthritis, diabetes, hereditary hemochromatosis, schizophrenia, multiple sclerosis, systemic lupus erythematosus, sarcoidosis, cirrhosis, myositis, psoriasis, nephritis, cancer, in particular neck or head cancer, IgA nephropathy, high blood pressure, Behcets disease, Gee-Heubner-Herter-Thaysen disease, myasthenia gravis, spondylarthropathia, tuberculosis, hypertrophic cardiomyopathy, Graves' disease, juvenile chronic arthritis, epilepsy, idiopathic generalized epilepsy or of juvenile epilepsy , Takayasu disease, multiple immunopathological diseases, for the susceptibility of leprosy, for the susceptibility of malaria and / or for the susceptibility
  • the nucleic acids listed in the appendix in accordance with SEQ ID-NO: 1 or SEQ ID-NO: 2 are also preferably used for the diagnosis of genetic and epigenetic parameters within the major histocompatibility complex (MHC). Furthermore, a method for the diagnosis of important genetic parameters within the MHC by analysis of cytosine methylations and single nucleotide polymorphisms in genomic DNA samples is described. This is done in the following steps:
  • a genomic DNA sample is chemically treated in such a way that at the 5 'position unmethylated cytosine bases are converted into uracil, thymine or another base which is unlike cytosine in terms of hybridization behavior.
  • the genomic DNA to be analyzed is preferably obtained from the usual sources for DNA, such as. B. cell lines, blood, sputum, stool, urine, brain spinal fluid, paraffin-embedded tissue, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lungs, chest or liver, histological slides and all possible combinations of these.
  • fragments are amplified from the chemically pretreated genomic DNA using primer oligonucleotides.
  • More than 10 different fragments that are 100-2000 base pairs long are preferably amplified.
  • the amplification is carried out by means of the polymerase chain reaction (PCR) through, preferably using a thermostable DNA polymerase.
  • PCR polymerase chain reaction
  • the amplification of several DNA sections is carried out in one reaction vessel.
  • the set of primer oligonucleotides comprises at least two oligonucleotides, the sequences of which are each reversely complementary or identical to a section of the base sequences according to SEQ ID-NO: 1 or SEQ ID-N0: 2 that is at least 18 base pairs long.
  • the primer oligonucleotides are preferably characterized in that they contain no CpG dinucleotide.
  • At least one primer is bound to a solid phase during the amplification.
  • oligonucleotides and / or PNA oligomer sequences are arranged on a flat solid phase in the form of a rectangular or hexagonal grid.
  • the solid phase surface preferably consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.
  • the amplificates are hybridized to a set of at least 10 oligonucleotide or PNA oligomer probes.
  • the oligonucleotides mentioned comprise at least one base sequence with a length of 13 nucleotides which are reverse complementary or identical to a section of the base sequences listed in the appendix which contains at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide viewed from the 5 'end of the 13 mer.
  • the PNA oligomers mentioned comprise at least one base sequence with a length of 9 nucleotides, which is reverse complementary or identical to a section of the base sequences according to SEQ ID-NO: 1 or SEQ ID-NO: 2, which contains at least one CpG dinucleotide ,
  • the cytosine of the CpG dinucleotide is the 4th to 6th nucleotide as seen from the 5 'end of the 9 mer.
  • the non-hybridized amplificates are removed.
  • the hybridized amplificates are detected.
  • markings attached to the amplificates can be identified at any position of the solid phase at which an oligonucleotide sequence is located.
  • the labels of the amplified products are fluorescent labels.
  • the labels of the amplificates are radionuclides.
  • the labels of the amplificates are detachable molecular fragments with a typical mass, which are detected in a mass spectrometer. It is preferred according to the invention that the amplified products, fragments of the amplified products or probes complementary to the amplified products are detected in the mass spectrometer.
  • the fragments generated have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the detection is carried out and visualized using matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electrospray mass spectrometry (ESI).
  • MALDI matrix-assisted laser desorption / ionization mass spectrometry
  • ESI electrospray mass spectrometry
  • the use of a method for diagnosing important genetic parameters within the MHC is preferred.
  • the present invention also relates to a kit consisting of a bisulfite-containing reagent, a set of primer oligonucleotides comprising at least two oligonucleotides, the sequences of which each have at least one 18 base pair long section of the base sequences according to SEQ ID-NO: 1 or SEQ ID-NO: 2 correspond to or are complementary to them for the preparation of the amplificates, oligonucleotides and / or PNA oligomers and instructions for carrying out and evaluating the described method.
  • the following example relates to a fragment of the HLA-A gene, in which a specific CG position is examined for methylation.
  • a genomic sequence is converted using bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis.
  • This converted DNA is used to detect methylated cytosines.
  • the cytosines of the HLA-A gene of length 3201 are examined.
  • a defined fragment of length 874 is amplified with the specific primers TTTGGTTTTGATTTAGATTTGG and AAATAAACTCTCTAACTACTC.
  • This amplificate serves as a sample which hybridizes to an oligonucleotide previously bound to a solid phase, for example TAGGTCGTTTATA, the cytosine to be detected being at position 487 of the amplificate.
  • the detection of the hybridization product is based on Cy3 and Cy5 fluorescent-labeled primers, which were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide occurs only if a methylated cytosine was present at this point in the bisulfite-treated DNA. The methylation status of the respective cytosine to be examined thus decides on the hybridization product.
  • Example 1 Performing the methylation analysis of the gene DAXX located in the MHC
  • FIG. 1 relates to a fragment of the DAXX gene in which a specific CG position is examined for methylation.
  • a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that all cytosines not methylated at the 5-position of the base are changed in such a way that a base which is different with regard to the base pairing behavior is formed, while the in the 5-position methylated cytosines remain unchanged.
  • bisulfite is used for the reaction, an addition takes place at the unmethylated cytosine bases.
  • a denaturing reagent or solvent and a radical scavenger must be present.
  • the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA is then preferably carried out.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase. In the present case, cytosines of the DAXX gene are examined.
  • a specific fragment of length 880 bp is amplified with the specific primer oligonucleotides TTAGGTTTTGGTTTGTTGATGAG and CCCTAACTCCTCTAAACCTCA.
  • This amplificate serves as a sample which hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, for example TAAAGAGGCGGATTAGTT, the cytosine to be detected being at position 142 of the amplificate.
  • the detection of the hybridization product is based on Cy3 and Cy5 fluorescence-labeled primer oligonucleotides that were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide only occurs if there is a methylated cytosine in the bisulfite-treated DNA at this point.
  • the methylation status of the individual to be examined is therefore decisive Cytosine via the hybridization product. In the present case, a non-methylated status is proven for the oligomer.
  • Example 2 Performing the methylation analysis of the RXRB gene located in the MHC
  • FIG. 2 relates to a fragment of the RXRB gene in which a specific CG position is examined for methylation.
  • a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that all cytosines that are not methylated at the 5-position of the base are modified in such a way that a base which is different with regard to the base pairing behavior is formed, -Position methylated cytosines remain unchanged.
  • bisulfite hydrogen sulfite, disulfite
  • an addition takes place at the unmethylated cytosine bases.
  • a denaturing reagent or solvent and a radical scavenger must be present. Subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases into uracil.
  • the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA is then preferably carried out.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase.
  • cytosines of the RXRB gene are examined.
  • a defined fragment with a length of 385 bp is amplified with the specific primer oligonucleotides ATATTGGTAAAGGTATTAGGG and ACTTAACTCAACTCTATACCTAC. This amplificate serves as a sample that is bound to a previously bound to a solid phase O CO IV) LO M
  • DJ rt DJ d et ß CO M li M 3 tr N 3 * P- ⁇ H rt ⁇ DJ iQ M fi? 3 cn DJ ⁇ -3 ⁇ s: 3- ⁇ cn li P- rt s Hi P- ⁇ P- • ⁇ M P- 3 ⁇ P- 3 ⁇ rt DJ ISI 3 0 P-
  • the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA is then preferably carried out.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase.
  • cytosines of the BAT5 gene are examined. For this purpose, a defined fragment with a length of 539 bp is amplified with the specific primer oligonucleotides AGAAGAGAATGTGGGTAGGA and AAAACCTACTTATCAAACCAAT.
  • This amplificate serves as a sample which hybridizes to oligonucleotides previously bound to a solid phase with the formation of a duplex structure, for example i) TGAGAAAGCGGTAAAGAG or ii) ATTTAAGGCGAGGGTAAA, the cytosine to be detected being for the oligomer TGAGAAAGCGGTAAAGAG at position 211 or for the oligomer PositionGTGGGATAAGA1GTAAGTAAGTAAGTAATAAGATAAGTAAGTAAGAAGGTAAGAGAAGAGAAGGTAAGAGTAAGTAAGTAAGAGAAGAGAAGGGAGAGAGAGAGAAA Amplificate is located.
  • the detection of the hybridization product is based on Cy3 and Cy5 fluorescence-labeled primer oligonucleotides that were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide only occurs if there is a methylated cytosine in the bisulfite-treated DNA at this point.
  • the methylation status of the respective cytosine to be examined thus decides on the hybridization product. In the present case, a partially ethylated status is shown for both oligomers in Figure A and a non-methylated status in Figure B.

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Abstract

L'invention concerne des acides nucléiques servant au diagnostic d'un jeu de paramètres génétiques à l'intérieur du complexe majeur d'histocompatibilité (CMH) et comprenant un segment identique ou complémentaire inverse, long d'au moins 20 paires de bases, d'un ADN génomique prétraité chimiquement. L'invention concerne également un jeu de sondes oligomériques (oligonucléotides ou oligomères PNA) servant à détecter l'état de méthylation de la cytosine dans des acides nucléiques. Ces sondes conviennent particulièrement au diagnostic de paramètres génétiques à l'intérieur du CMH.
EP01955494A 2000-06-30 2001-06-29 Diagnostic de parametres genetiques importants a l'interieur du cmh Withdrawn EP1358351A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10032529A DE10032529A1 (de) 2000-06-30 2000-06-30 Diagnose von bedeutenden genetischen Parametern innerhalb des Major Histocompatibility Complex (MHC)
DE10032529 2000-06-30
PCT/IB2001/001501 WO2002000932A2 (fr) 2000-06-30 2001-06-29 Diagnostic de parametres genetiques importants a l'interieur du cmh

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AU (1) AU2001277653A1 (fr)
DE (1) DE10032529A1 (fr)
WO (1) WO2002000932A2 (fr)

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DE10032529A1 (de) 2002-02-07
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JP2004511217A (ja) 2004-04-15
WO2002000932A3 (fr) 2003-09-04

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