WO2005010183A1 - 薬剤起因性顆粒球減少症発症リスク判定法 - Google Patents
薬剤起因性顆粒球減少症発症リスク判定法 Download PDFInfo
- Publication number
- WO2005010183A1 WO2005010183A1 PCT/JP2004/010722 JP2004010722W WO2005010183A1 WO 2005010183 A1 WO2005010183 A1 WO 2005010183A1 JP 2004010722 W JP2004010722 W JP 2004010722W WO 2005010183 A1 WO2005010183 A1 WO 2005010183A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- polymorphism
- oligonucleotide
- human
- coding region
- Prior art date
Links
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
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- 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
- C12Q2600/156—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
- C12Q2600/172—Haplotypes
Definitions
- the present invention provides a method for determining the risk of developing drug-induced granulocytopenia using the presence of a gene polymorphism in the human insulin receptor substrate-12 gene as an index,
- the present invention relates to detection methods, oligonucleotides used in those methods, and determination and / or detection kits.
- the main drugs that cause this granulocytopenia include analgesics and antipyretics (aminopyrine), antibiotics (chloromycetin), antithyroid drugs (mercazole), anticonvulsants, antidiabetic drugs, and diuretics.
- analgesics and antipyretics aminopyrine
- antibiotics chloromycetin
- antithyroid drugs mercazole
- anticonvulsants antidiabetic drugs
- diuretics diuretics.
- vesnarinone Another drug is vesnarinone, which has a PDE3 inhibitory effect and an effect on K channels.
- This drug is effective as a cardiotonic drug with a low incidence of cardiac arrhythmias with low incidence of arrhythmias (onset of heart failure, hospitalization, etc.). Its use is severely restricted due to side effects that cause the disease.
- SNPs single nucleotide polymorphisms
- the SNP has been shown to be useful in human genome analysis relating to common diseases, drug responses, and the like (see Non-Patent Documents 1, 2, and 3).
- haplotype analysis using a plurality of SNPs is useful for analyzing disease susceptibility in genetically complicated diseases (see Non-Patent Documents 4 and 5).
- diseases such as Alzheimer's disease and hypertension have been intensively analyzed by this method ieunemaitre, X., et a 1., Am. J. Hum. Genet., 60, 1448-1460 ( 1997); Martin, ER, Am. J. Hum. Genet., 67, 383-394 (2000)).
- Non-Patent Document 1 Brookes, AJ, "The essence of SNPs", Gene, USA, (19 99), 234, 177-186
- Non-Special Publication No. 2 Cargill, M, et al., "Characterization of single-nucleotide polymorphisms in coding regions of human genes", Nature Genet., USA, (1999), 22, 231-238
- Non-Special Publication No. 3 Evans, W.E., & Relling, M.V., Pharmacogenomics: translating functional genomics into rational therapeutics, Science, USA, (1999), 286, 487-491
- Non-Patent Document 4 Stephens, JC, et al., "Dating the origin of the CCR5 — Delta32 AIDS—resistance allele by the coalescence of haplotypes, Am. J. Hum.Genet., USA, (1998), 62, 1507 —1515
- Non-special language reference 5 Tishkoff, SA, et al., "The accuracy of statistical met hods for estimation of haplotype frequencies: an example from the CD4 locus", Am. J. Hum. Genet., USA, (2000), 67, 518—522 DISCLOSURE OF THE INVENTION
- the present invention provides a means for determining the risk of developing drug-induced granulocytopenia using the presence of the human 'insulin receptor substrate 12 gene as an index or a means for detecting a gene polymorphism that is an index for the risk determination.
- the main challenge is to provide
- the present inventors firstly, as polymorphism analysis genes, cytoforce-in related, MHC region, G-CSF related, TNF- ⁇ related, NF ⁇ related, cAMP related, We selected 115 candidate genes, including those related to K-channel, and searched SNPs of these candidate genes from the Japanese polymorphism database, and selected 188 candidate SNPs.
- the frequency of the SNPs appearing in the sample genomic DNA of two groups, a patient group with onset of granulocytopenia and a group of patients without onset, which was developed by administration of the specific drug was determined.
- the SNPs strength, the insulin receptor substrate-2 gene (Insulin receptor substrate 2: IRS-2) (J-SNP ID: IMS-JST040476) showing the statistically most significant difference between the above two groups , Called "IRS_2 gene" confirmed.
- the present inventors have further studied the relationship between the polymorphism of the human IRS-2 gene and drug-induced granulocytopenia. A total of six closely related SNPs of the human IRS-2 gene were identified.
- the present invention provides a method for determining the presence of the risk of developing drug-induced granulocytopenia shown in the following (1)-(19) or a method for detecting a genetic polymorphism marker serving as an index for the risk determination, and And a kit for use in the method.
- Wild type (A) at position 15870 from the translation initiation codon of the coding region is mutated to G. Polymorphism
- Gene polymorphisms are detected by direct nucleotide sequencing, allele-specific oligonucleotide (ASO) _dot plot analysis, single-base primer extension, PCR-single-stranded higher-order structural polymorphism (SSCP) analysis, PCR-restriction fragment length polymorphism (RFLP) analysis, invader method, quantitative real-time PCR detection method, and gene polymorphism detection method using mass spectrometer (mass array)
- ASO allele-specific oligonucleotide
- SSCP PCR-single-stranded higher-order structural polymorphism
- RFLP PCR-restriction fragment length polymorphism
- PCR-restriction fragment length polymorphism (RFLP) analysis was performed using the restriction enzyme Afa I to detect the A-G mutation at position 29793 from the translation initiation codon of the coding region of the human IRS2 gene.
- oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to the human IRS2 gene, wherein the oligonucleotide is selected from the group consisting of the following (a)-(f):
- oligonucleotide is selected from the group consisting of the following (a) _ (d) and (f);
- Drug-induced granulocytopenia risk assessment including the oligonucleotide according to (9) above as a primer for detecting a polymorphism in the human IRS2 gene or a probe for detecting a polymorphism in the human IRS2 gene Kit.
- a human IR comprising the oligonucleotide according to (9) (e) above and a restriction enzyme Afa I
- the risk of developing drug-induced granulocytopenia including the oligonucleotide according to (9) above as a primer for detecting a polymorphism in the human IRS2 gene or a probe for detecting a polymorphism in the human IRS2 gene.
- a method for determining the risk of developing drug-induced granulocytopenia in humans a method for detecting a genetic polymorphism that serves as an index for the above-described determination, a kit therefor, and a mutation used therein
- primers and probes for detection and genes associated with risk factors for developing drug-induced granulocytopenia in humans. These are used to detect and determine the risk of developing human drug-induced granulocytopenia, especially for drugs for which drug-induced side effects of drug-induced granulocytopenia (including agranulocytosis) have been reported. It is useful for examining and determining the risk of developing granulocytopenia before administration.
- FIG. 1 is a schematic diagram showing the structure of a human IRS-2 gene and the locations of gene polymorphisms.
- genomic sequence of the human IRS-2 gene shown in the present specification is the full-length sequence reported as GenBank Accession Number: AL162497 by Mohammadi (M.) of Sanger Center. Included in sequences of 143, 409 bases (bp).
- the IRS-2 gene estimated from the genomic sequence obtained based on the sequence information of IRS-2 mRNA obtained from GenBank (accession number XM 007095) and the sequence information of AL162497 described above consists of two ethasons.
- the total length of the gene including the intron is 32,730 bp.
- This gene corresponds to 93673-126402 bp in the system IJ of AL162497.
- the outline of the structure of the gene is shown in FIG. In Figure 1, “ ⁇ ⁇ 1” and “ ⁇ .2” indicate two exons. Abbreviations indicated by arrows indicate the following mutations (SNPs). These are all synonymous substitutions (those that do not change the sequence of the protein product).
- the location number of the SNP shown in the text or the figure is shown as the location number from the position ⁇ of the translation initiation codon ATG of the coding region encoding the protein.
- AT_2510del deletion of AT at position 2510 upstream from translation initiation codon of human IRS-2 gene coding region
- A_l 164C mutation from A to C at position 1164 upstream from the translation initiation codon of the coding region of the human IRS-2 gene;
- A15870G mutation from A to G at position 15870 from the translation initiation codon of the coding region of the human IRS-2 gene
- the term "gene” refers not only to double-stranded DNA but also to each of the constituents thereof. Includes single-stranded DNA (sense strand and antisense strand). That is, unless otherwise specified, the gene of the present invention (DNA) is a double-stranded DNA containing human genomic DNA, a single-stranded DNA containing cDNA (sense strand), and a single-stranded DNA having a sequence complementary to the sense strand. Includes DNA and their fragments.
- the gene (DNA) can include a regulatory region, a coding region, exons, and introns. Polynucleotides include RNA and DNA.
- DNA includes cDNA, genomic DNA, and synthetic DNA.
- Polypeptides include fragments, homologs, derivatives and variants thereof.
- variants do not substantially alter the function of naturally occurring allelic variants, non-naturally occurring variants, modified (deletion, substitution, addition and insertion) variants and encoded polypeptides. Stands for polynucleotide sequence.
- the alteration in the amino acid sequence may naturally occur due to, for example, mutation or post-translational modification, and can be artificially performed using a naturally-occurring gene.
- SNP Single nucleotide polymoi "phism: -nucleotide polymorphism
- a haplotype is a mutation of a single nucleotide nucleic acid (SNP), which is represented by the type and number of alleles at multiple mutation sites in a continuous gene region or a group of genes. ) Indicates the type.
- the present invention relates to polymorphisms containing mutations at specific positions in the human IRS-2 gene (the entire IRS-2 gene including a promoter region involved in transcriptional regulation), particularly SNP or SNPs-powered S-human drug-induced granules. Comprehensively based on the discovery that the risk of developing drug-induced granulocytopenia can be determined (predictive diagnosis) by detecting SNP at a specific location as a genetic polymorphism marker and strongly correlating with cytopenia. ing.
- the determination method of the present invention relates to detecting a polymorphism of the human IRS-2 gene in a sample (derived from a subject), that is, detecting SNPs or SNPs of the human IRS-2 gene.
- the SNPs detected and analyzed by the method of the present invention include the aforementioned C-1587A, AT-2510del, and A-1164C. , A15870G, A29793G and C31532del are included.
- the location on the IRS-2 gene where they exist is as shown in FIG. However, sandwiched between nucleic acids
- the number of the SNPs present indicates the number of the SNPs from ATG of the translation initiation codon of the coding region of the coding region encoding the protein of the IRS-2 gene.
- polymorphisms (SNPs and haplotypes) of the human IRS-2 gene can be detected, whereby the function elucidation, comprehension, diagnosis and prevention of the onset of drug-induced granulocytopenia in humans can be detected.
- by determining a patient at risk of developing drug-induced granulocytopenia, and avoiding drug administration to the patient the onset of drug-induced granulocytopenia is prevented. Can be prevented.
- countermeasures against side effects can be effectively performed by performing frequent tests in consideration of the occurrence of side effects at the time of drug administration in conjunction with treatment or treatment.
- the method of the present invention detects the genetic polymorphism of the human IRS-2 gene in a subject, and uses this as an index to determine the presence of a risk of drug-induced granulocytopenia.
- the genomic sequence of the human IRS-2 gene or its complementary strand was prepared from the subject, and the genomic sequence or its complementary DNA sequence was determined as necessary. Thereafter, the detection is performed by detecting the gene polymorphism.
- a human IRS-2 gene derived from a subject is prepared as a specimen. Specific examples of the gene having the polymorphism (SNPs) are as described above. The gene also includes a complementary strand of the DNA sequence of the human IRS- 12 gene exemplified above.
- the human IRS-2 gene having a genetic polymorphism or its complementary chain can be easily obtained by a general genetic engineering technique based on the specific sequence information of the human IRS-2 gene disclosed herein. [Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); See Seismic Chemistry Laboratory Course “Gene Research Methods I, II, III”, edited by The Biochemical Society of Japan (1986), etc.).
- cDNA or genomic DNA is extracted from a human drug-induced granulocytopenia patient having the human IRS-2 gene SNPs according to a conventional method, and the specific mutation of the human IRS-2 gene is identified.
- Conventional methods using suitable probes, restriction enzymes, antibodies and the like [Proc. Nat 1. Acad. Sci., USA, 78, 6613 (1981); Science, 222, 778 (1983), etc.
- the desired clone can be selected in accordance with the above-mentioned method to prepare the genome system J of the desired IRS-2 gene.
- examples of the source of cDNA or genomic DNA include various cells and tissues having the IRS-2 gene (SNPs), and cultured cells derived therefrom.
- blood such as serum and plasma, body fluids such as saliva, lymph, airway mucus, urine, and semen can be exemplified.
- the source material used as a specimen is DNA or genomic DNA from a patient before administration of a drug to a human (particularly before administration of a drug for which drug-induced granulocytopenia has been reported in the past).
- Isolation of RNA from these source materials, isolation and purification of mRNA, acquisition of cDNA, cloning thereof, and the like can all be carried out in accordance with ordinary methods.
- cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries commercially available from Clontech Lab. Inc. and the like can also be used.
- the method for screening a desired gene from a cDNA library is also not particularly limited, and can be according to a usual method. Specifically, a probe containing a mutated portion that can selectively bind to the DNA sequence of the target SNPs is prepared and used for plaque hybridization, colony hybridization, or the like. These may be implemented in combination.
- a forward primer and a reverse primer set based on the nucleotide sequence information of the desired human IRS-2 gene can be used. These can be synthesized according to a conventional method, for example, using an automatic synthesizer.
- the screening probe is usually a labeled probe, but may be an unlabeled probe as long as it can directly or indirectly specifically bind to the labeled ligand. Labeling agents and labeling methods for probes and ligands are already well known in the art.
- radiolabeling agents examples include radiolabeling agents, biotin, fluorescent dyes, chemiluminescent agents, enzymes such as luciferase, antibodies that can be incorporated by known methods such as nick 'translation, random' priming, and kinase treatment. Can be exemplified.
- the extracted gene or mRNA can be amplified by a gene amplification method.
- detection in the detection method of the present invention can be performed more easily and with higher accuracy.
- the gene amplification method include the PCR method (Saiki, RK, Bugawan, TL, et al., Nature, 324, 163-166 (1986)) and the NASBA method (Comptom, J., Nature, 650, 91- 92 (1991)), the TMA method (Kacian, DL, and Fultz, T.J., U.S. Pat.No. 5,399,491 (1995)), the SDA method (Walker, GT, Little, III.C., et. al., Pro Natl. Acad. Sci., USA, 89, 392-396 (1992)).
- the gene fragment amplified by the PCR method or the like may be isolated and purified by a conventional method, for example, gel electrophoresis, or may be purified by a column.
- the confirmation can be made, for example, by the mass storage method.
- the gene amplified by these methods is used for detection of the human IRS-2 gene (SNPs) according to the present invention, depending on the characteristics of the amplified product.
- the presence or absence of the polymorphism in the sample is then detected.
- This detection can be specifically carried out, for example, according to various methods shown in the following (1)-(8).
- Detection of the IRS-2 gene is commonly used to determine the nucleotide sequence of this type of gene, for example, the dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467). (1977)) and the direct nucleotide sequencing method such as the maxam-one-ginorate method [Methods in Enzymology, 65, 499 (1980)]. It can also be carried out according to a method combining this method and a DNA amplification method such as a PCR method. In particular, from the viewpoint of simple and easy use of a small amount of DNA sample and high-sensitivity and high-accuracy detection, it is preferable to use a method combining a PCR method or a DNA amplification method according to the method.
- This preferred method is basically, for example, by cloning a gene fragment amplified by a PCR method or a purified product thereof into a plasmid, and then directly sequencing the nucleotide sequence according to the dideoxy method, the maxamugilbert method or the like. Can be implemented. Also, it can be conveniently carried out by determining the nucleotide sequence using a commercially available sequence kit or the like. By virtue of the presence of the mutation at the aforementioned specific site of the human IRS-2 gene, None can be detected.
- the DNA fragment to be amplified by the PCR method as a specimen is not particularly limited as long as it contains at least one of the specific sites where the above-mentioned mutation is supposed to exist. Absent. Usually, it should have a length of about 50 to several thousand bases, preferably 50 to several hundred bases.
- An alternative method of detecting the IRS-2 gene is the allele-specific oligonucleotide (AS ⁇ ) -dot blot method (Conner, BJ, et al., Proc. Natl. Acad. Sci., USA, 80, 278-282). (1983)).
- the method uses, for example, a forward 'primer' and a 'reverse' primer designed to sandwich the target SNP, and a DNA fragment that hybridizes to an allele-specific oligonucleotide 'probe to a PCR-amplified gene fragment is obtained. This can be done by dot 'plot analysis. By virtue, it can be determined whether SNPs are present in the fragment.
- Detection of the IRS-2 gene can also be performed by a single base extension method such as a snapshot method, a pyrosequence method, or a point mutation detection method disclosed in JP-A-2000-279197.
- a probe set to correspond to the base immediately before or several bases before the target mutation (SNP), that is, its 3 ′ end is located one base upstream or near the target mutation. Anneal the set probe to the DNA sample.
- SNP target mutation
- Each method can be carried out using a commercially available kit for detecting SNPs and software attached to the kit.
- the snapshot method can be carried out using ABI PRISM SNaPshot ddNTP Primer Extension Kit (manufactured by ABI Biosystems). SNPs can be detected and analyzed for fluorescent fragments generated after the reaction using ABI PRISM310 / 377/3100/3700 DNA Analyzer (all manufactured by ABI Biosystems) and GeneScan software.
- the pyrosequencing method can be performed, for example, as follows. That is, genomic DNA is isolated from a blood sample or the like by a conventional method, and mutation is detected using biotin-labeled primers. PCR amplification of several tens of hundreds of bases is performed, and single-stranded DNA is purified using magnet beads, and this purified DNA is used as a sample. The sample is annealed with a primer set to sequence from a few bases upstream of the desired mutation, and then dNTPs are added to the device one by one according to the sequence near the mutation input to the software.
- Pyrophosphate is generated when DNA polymerase base-extends, and this PPi is converted back to ATP by sulfurylase, and this is used as a substrate for luciferase and chemiluminescent using a luminescence detector, CCD camera, etc. Is detected.
- a luminescence detector CCD camera, etc.
- reagents and devices include those usually used, for example, DNA polymerase, ATP-sulfurylase, luciferase and a mixture of four kinds of enzymes of apyrase, luciferin and APS (adenosine ⁇ sulfate phosphate).
- reagents such as dATP (doxyadenosine triphosphate), commercially available SNP Reagent Kits (manufactured by Pyrosequencing AB), and dNTP consisting of dCTP, dGTP and dTTP, and automated DNA sequence analysis Using the PSQ96 system (Pyrosequencing AB) and SNP software (Pyrosequencing AB) for its use.
- dATP dioxyadenosine triphosphate
- SNP Reagent Kits manufactured by Pyrosequencing AB
- dNTP consisting of dCTP, dGTP and dTTP
- automated DNA sequence analysis Using the PSQ96 system (Pyrosequencing AB) and SNP software (Pyrosequencing AB) for its use.
- the pyrosequencing method is, for example, as described in US Patent No. 6,159,693, after isolating a nucleic acid, amplifying the nucleic acid, purifying the amplified PCR product, and then reading the READITTM System (Promega Corporation).
- the reaction can also be carried out by reacting pyrophosphoric acid with this and analyzing the obtained data.
- Excel analysis using a commercially available READIT technology (manufactured by Promega Corporation) can be adopted.
- the detection method of the present invention includes a PCR-SSCP method in which a PCR amplification product (single-stranded DNA) is subjected to non-denaturing polyacrylamide gel electrophoresis, and the presence or absence of a single base mutation is determined based on the difference in mobility.
- a PCR amplification product single-stranded DNA
- non-denaturing polyacrylamide gel electrophoresis non-denaturing polyacrylamide gel electrophoresis
- PCR—Nagaro H ⁇ ⁇ ⁇ (RFLP) In the detection of SNPs or haplotypes of the human IRS-2 gene of the present invention, for example, when the nucleic acid sequence containing the mutation to be detected contains a restriction enzyme recognition site, the detection is performed by restriction fragment length polymorphism. It can also be performed by an analytical method (RFLP method: Botstein, DR, et al., Am. J. Hum. Gen., 32, 314-331 (1980)).
- the nucleic acid sequence at position 29793 from the translation initiation codon of the coding region of the protein present in the second exon of the human IRS-2 gene is wild type (A) or mutant type (G).
- A wild type
- G mutant type
- a restriction enzyme capable of recognizing the sequence before and after the mutation site is used.
- the enzyme used in the powerful RFLP method may be any of various known restriction enzymes capable of recognizing the sequence before and after the target mutation site. A specific example thereof is Afa I.
- the RFLP method is more preferably carried out on a sample DNA which has been prepared and concentrated in a large amount after the sample DNA has been amplified'prepared by the PCR-RFLP method, that is, the PCR method or a modification thereof in advance. Can be by way. By virtue of this, the presence or absence of a mutation can be detected as the presence or absence of a specific cleavage site.
- Detection of the SNP of the human IRS-2 gene by the PCR-RFLP method is more specifically performed, for example, according to the following method. That is, first, genomic DNA of the human IRS-2 gene is extracted from a human biological sample, and a DNA fragment in a region containing a mutation site of the gene is amplified to obtain a large and concentrated sample of the sample.
- the forward primer and / or the reverse primer used herein include those whose sequence does not completely match the genome sequence, and preferably those into which a sequence for introducing a restriction enzyme recognition site has been introduced. It is.
- the amplified DNA sample is digested with a specific restriction enzyme (that is, an enzyme that can digest only either the wild-type or mutant type), and the DNA cleavage mode (presence / absence of cleavage, base length of the cleavage fragment). Etc.) according to the usual method.
- a specific restriction enzyme that is, an enzyme that can digest only either the wild-type or mutant type
- the DNA cleavage mode Presence / absence of cleavage, base length of the cleavage fragment. Etc.
- the base of the human IRS-2 gene J29793-29796
- GTAC specific cleavage site
- 16 ⁇ Invader method SNPs of the IRS-2 gene can also be detected by the Invader method.
- the following documents can be referred to for implementing the invader method.
- This method is a method that can be performed without amplifying the target DNA in advance to analyze SNPs of genomic DNA.
- the method is performed as follows.
- genomic DNA is first isolated.
- a ⁇ flap having a length of 15 to 50 bases and a nucleic acid to be detected are arranged at the 3 'end of the 5' flap, and the target genome DN except for the mutant nucleic acid
- a first target probe consisting of an oligonucleotide of 30 to several hundred bases synthesized to complement A and a nucleic acid complementary to the nucleic acid to be detected are placed at the 3 'end Invader oligonucleotide consisting of oligonucleotides of 15 to several tens bases in length, synthesized with a probe, for example, using an automatic synthesizer
- genomic DNA in the sample contains the desired mutant nucleic acid (SNP)
- SNP mutant nucleic acid
- a first reaction occurs that releases the 5 'flap with the mutant nucleic acid at the end. If the genomic DNA in the sample has a mutated nucleic acid sequence, the enzyme does not cause cleavage.
- the 5 'flap released from the first probe cleaved by the enzyme binds complementarily to the fluorescence resonance energy transfer (FRET) probe as a target, and the 3' end of the 5 'flap is located inside the FRET probe. Invasion. Similarly, a reaction by the enzyme occurs, releasing the fluorescent dye.
- FRET fluorescence resonance energy transfer
- Each FRET probe used in this second reaction is independent of the target to be detected and is constructed to include essentially two elements:
- the reporter fluorescent dye When the reporter fluorescent dye is bound to the same probe as the quencher-fluorescent dye, the fluorescence intensity of the reporter fluorescent dye is suppressed by fluorescence resonance energy transfer. When not bound to the same probe as the dye, the fluorescence intensity is not suppressed.
- the 5 'flap force released from the cleaved first probe When hybridized to a FRET probe, it acts as an Invader oligonucleotide in a second reaction, producing an invasion complex that is recognized by the enzyme. By force, the cleavage force of the FRET probe by the enzyme separates the two fluorescent dyes and produces a detectable fluorescent signal.
- the signal can be read, for example, with a standard fluorescence microtiter plate reader, whereby the presence or absence or the haplotype of the desired SNPs can be detected.
- the combination of the first and second reactions can amplify the signal from 1 to 1 ⁇ 10 6 fold.
- the presence or absence of SNP can be detected (typed) by using two types of fret probes with different fluorescent dyes.
- Detection of the human IRS-2 gene polymorphism can also be easily performed by a quantitative real-time PCR detection method (TaqMan method).
- the method can be performed, for example, as follows. That is, first, a DNA fragment containing a nucleic acid site to be detected for the presence or absence of a target mutation is prepared as, for example, a 15-39 nucleotide base primer and a reverse primer. However, the forward primer and the reverse primer should be prepared so as not to contain the target mutation. Next, a probe is prepared, which is an oligonucleotide having a base sequence of, for example, 1550 bases, in which a reporter fluorescent dye and a quencher fluorescent dye are bound. However, as the base sequence of the probe, a combination in which the region where the forward primer hybridizes and the region where the probe hybridizes does not overlap each other is selected.
- the probe is prepared so as to have a sequence complementary to an allele-specific sequence for detecting the presence or absence of a target single-base nucleic acid mutation.
- a desired DNA fragment of the IRS-2 gene to be measured in the sample is amplified by PCR, The fluorescence from the reaction solution is measured in real time. By virtue of this, the presence or absence of the mutation can be detected.
- the detection (typing) of the presence or absence of SNP can also be performed by using two types of probes having different fluorescent dyes.
- a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable.
- FAM 6-carboxy-fluorescein
- Carboxy-tetramethyl-rhodamine is preferred.
- These fluorescent dyes are known and can be used because they are contained in a commercially available kit for real-time detection PCR.
- the binding positions of the reporter fluorescent dye and the quencher monofluorescent dye are not particularly limited. Generally, the reporter fluorescent dye is bonded to one end (preferably, the ⁇ end) of the oligonucleotide portion of the probe, and the quencher monofluorescent dye is bonded to the other end.
- the TaqMan method itself is known, and devices and kits for it are also commercially available. Therefore, such commercially available devices and kits can be used in the present invention.
- the force according to the method described in Patent No. 2,825,976, the ABI PRISM 7700 sequencing system manufactured by PE Biosystems' user manual may be followed.
- the mass array method is a method for detecting a difference in mass caused by a polymorphism. Specifically, after the region containing the polymorphism to be detected is amplified by PCR, the extension primer is hybridized immediately before the SNP position, and the reaction solution containing the ddNTPZdNTP mixture, such as ddATP, dCTP, dGTP and dTTP By performing an extension reaction using the reaction solution, fragments with different lengths depending on the SNP are generated.
- the reaction solution containing the ddNTPZdNTP mixture such as dddATP, dCTP, dGTP and dTTP
- the correspondence between the mass number and the genotype can be analyzed (Pusch, W., Wurmbach, JH., Thiele, ⁇ ., Kostrzewa, M., MALDI — TOF mass spectrometry—based SNP genotyping, Pharmacogenomics, 3 (4): 537-48 (2002)).
- the method can be used, for example, in Sequenom Mass ARRAY High It can be easily performed using a throughput SNP analysis system (http: ZZwww.sequenom.com/Files/applications/hme-assay.html).
- Detection of SNPs of the human IRS-2 gene can be carried out by the following various methods conventionally known as a method for determining the nucleotide sequence of DNA and a method for detecting mutations.
- a probe for each mutation is immobilized on a carrier, and a sample (gene amplification product) is hybridized to the carrier to determine a difference in hybridization efficiency depending on the presence or absence of a mismatch.
- PCR-DGGE a method that compensates for the shortcomings of detection when multiple base substitutions, deletions, additions, and insertions are made by connecting a region with high GC content to the DNA fragment from which the mutant nucleic acid is to be detected It is.
- the method particularly requires a step of adding a GC clamp to a DNA fragment to be subjected to mutation detection.
- SNPs polymorphisms
- haplotypes of the human IRS-2 gene can be detected.
- the risk of onset of drug-induced granulocytopenia according to the present invention is determined by using the presence of the human IRS-2 gene polymorphism detected as described above as an index for a sample in which the presence of the polymorphism is confirmed. Then, the sample is determined to have a high risk.
- Patients determined to have a high risk in this manner can confirm the risk of causing drug-induced condyleopenia before bowel administration before administration of the drug, and therefore, the presence of The onset of hypotension can be prevented beforehand.
- detection of SNPs of the human IRS-2 gene according to the present invention is effective for detecting the presence of a drug-induced risk factor for the development of granulocytopenia in humans. Risk factors for the development of granulocytopenia can be detected.
- the present invention provides a method for detecting the risk of developing drug-induced granulocytopenia in a subject to be subjected to the detection of human IRS-2 gene SNPs, using the presence of a human IRS-2 gene polymorphism as an index. And a method for detecting a gene polymorphism of the gene for the determination.
- the present invention also provides an oligonucleotide as a primer or probe for detecting a gene polymorphism used in the determination (detection) method of the present invention employing a PCR method.
- the oligonucleotide is not particularly limited as long as it can specifically amplify a specific sequence portion including a mutated portion (SNPs) of the human IRS-2 gene.
- the oligonucleotide can be appropriately synthesized and constructed according to a conventional method based on the sequence information of the human IRS-2 gene.
- the synthesis can be performed by a general chemical synthesis method such as a phosphoramidite method or a phosphotriester method, or a commercially available automatic oligonucleotide synthesizer such as (Pharmacia LKB Gene (Assembler Plus: manufactured by Pharmacia) can also be used.
- the double-stranded fragment is synthesized by synthesizing a chemically synthesized single-stranded product and its complementary chain and annealing them under appropriate conditions, or by using an appropriate primer sequence and DNA polymerase. It can be obtained by adding a complementary strand to the main strand product.
- the oligonucleotide used as the probe or the primer include a partial oligonucleotide corresponding to a DNA fragment set to contain a mutation of the human IRS-2 gene, and at least 10 However, those having usually about 10 to 35 consecutive bases can be exemplified.
- the primer pair can be one having two oligonucleotide sequences designed and synthesized so as to sandwich the SNP in the human IRS-2 gene (genome system 1J).
- a positive clone itself can be used as the oligonucleotide used as a probe.
- Preferable examples of the oligonucleotide used as the probe or primer include mutation from C to A at position 4587 upstream of the translation initiation codon of the coding region of human IRS-2 gene (C-4587A), human Mutation of AT at position 2510 upstream from the translation initiation codon of the coding region of the IRS-2 gene (AT_2510del), mutation from A to C at position 1164 upstream of the translation initiation codon of the coding region of the human IRS-2 gene (AT_2510del) A—1164C), a mutation from A to G at position 15870 from the translation initiation codon of the coding region of the human IRS-2 gene (A15870G), and A from position 29793 from the translation initiation codon of the coding region of the human IRS-2 gene.
- the gene-specific probe of the present invention may be any as long as it can detect the level shift force of C_4587A, AT-2510del, A-1164C, A15870G, A29793G, and C31532del.
- the determination (detection) method of the present invention can be carried out more easily by using a reagent kit for detecting SNPs of the human IRS-2 gene in a sample.
- the present invention also provides such a determination kit.
- kits of the present invention comprises at least a part or all of the nucleotide sequence that is the DNA fragment of the six SNPs of the human IRS-2 gene or its complementary nucleotide sequence, or one nucleotide before or at the mutation site. Contains a hybridizing DNA fragment as an essential component in the sequence consisting of the base sequence.
- Another one of the kit of the present invention contains a restriction enzyme that recognizes a nucleic acid sequence of several bases including the above mutation site, for example, Afa I as an essential component.
- kits of the present invention examples include a labeling agent and reagents essential for the PCR method (eg, Taq DNA polymerase, deoxynucleotide triphosphate, primers for DNA amplification, etc.).
- the labeling agent include a chemically modified substance such as a radioisotope, a luminescent substance, and a fluorescent substance.
- the DNA fragment itself may be conjugated with the labeling agent in advance.
- the kit may contain a reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., which are suitable for the convenience of performing the measurement.
- a polymorphism of a gene that may cause drug-induced granulocytopenia in humans is detected, and using this as an index, Methods for assessing the risk of granulocytopenia, especially prior to the administration of drugs for which drug-induced side effects of granulocytopenia (including agranulocytosis) have been reported by administration of drugs such as vesnarinone And a diagnostic agent and a diagnostic kit used in the method for detecting the risk of developing granulocytopenia.
- Vesnarinone is indicated in Japan for chronic heart failure (mild or moderate), but after administration of the drug, leukopenia, granulocytopenia, and agranulocytosis have been reported as drug-induced side effects after administration of the drug. Therefore, in drug administration, it is necessary to observe these side effects and conduct frequent granulocyte tests.
- the target patients were classified into two groups, a patient group with onset of granulocytopenia and a patient group without onset, according to the following criteria.
- Each of these groups was further divided into two groups according to gender, to make a total of four groups. 13 patients with granulocytopenia (Group A), 17 women with onset (Group B), 33 non-onset men (Group C), and 21 non-onset women (Group D) .
- 115 candidate genes were selected from genes related to cytokinin, MHC region, G-CSF, TNF- ⁇ , NF- ⁇ , cAMP and potassium 'channel.
- Japanese polymorphism database jSNP: http: Z snp. Ims. U— tokyo. Ac. Jp / index _ ja.html ) We searched for SNPs of these candidate genes and selected 188 candidate SNPs.
- SNP analysis was performed using the invader method.
- the invader method was carried out with reference to the following documents (1) and (2).
- a primer set to amplify each SNP region is set based on the genomic DNA sequence information including the SNP searched by JSNP, and each primer is synthesized. did.
- Atsey reagent for determining gene polymorphisms of candidate SNPs was prepared by a usual method based on genomic DNA sequence information including SNPs searched by JSNP. Each PCR reaction was performed using lng genomic DNA as type III. The reaction solution of 15 zL was prepared by adding the PCR reaction buffer attached to dNT Ps (0.25 mM) and TaKaRa Ex Taq (Takara) to 1/10 volume of the total reaction volume, each of the forward primer and reverse primer. Set (130 nM each) and TaKaRa Ex Taq (Takara) (0.5 U). Each sample was amplified by DNA Engine PTC_0200 (MJ Research).
- the Invader-1 'Atsey reaction was carried out by mixing the obtained PCR product with a 10-1000-fold dilution and the Inbeta-1'Atsey reagent.
- the 15 zL reaction mixture was prepared using 5.5 X Invader buffer (2.75 zL), lOX Bioplex FRET Probe Mix (0.75 ⁇ L), CI eavase VIII enzyme (200 ngZ ⁇ U (1 ⁇ U , PPI Mix (3 ⁇ L), and PCR product dilution (7. The reaction was performed at 62 ° C for 60-120 minutes.
- the genotype was determined based on the fluorescence intensities of the two colors detected as a result of the Invader-Atsey reaction. Intensely, Invader'Atsusei genotyped 188 SNPs present in 115 genes in the subject patients.
- IRS-2 insulin receptor substrate protein family
- IRSs insulin receptor substrate protein family
- Activation of the tyrosine kinase of the insulin receptor results in tyrosine phosphorylation.
- Phosphorylated IRSs activate PI-3 kinase, promote the transport of glucose transporter 4 (GLUT-4) from the cytoplasm to the cell membrane, and exert an insulin action that promotes glucose uptake. It has been known. Therefore, the human IRS-2 gene and granulopenia caused by vesnarinone To further investigate the association, a polymorphism analysis test of the human IRS-2 gene was performed.
- the genomic sequence containing the obtained IRS-2 gene was obtained from GenBank (accession number AL162497, full length 143409 bp). Detailed comparison between the sequence of human IRS-2 mRNA and the genomic sequence containing the IRS-2 gene was performed to estimate the gene structure of human IRS-2. However, genomic sequences obtained in order to unify the gene direction from the 5 'side to the 3' side were compared using their complementary strands.
- the human IRS-2 gene is composed of two ethasons, and that the length of this gene including introns is 32730 bp.
- genomic samples of 12 cases of granulocytopenia and 12 cases of non-granulocytopenia in the subject patients of Example 1 were used.
- Each PCR reaction was performed using 5 ng of genomic DNA. 10 ⁇ L of the reaction solution was dNT Ps (l. 25 mM), magnesium chloride (3.9 mM), ammonium sulfate (16.6 mM), Tris-HCl (67 mM, pH 8.8), and j3-mercaptoethanol (10 mM). , Each set of forward primer and reverse primer (1.25 mM), and TaKaRa Ex Taq (Takara) (0.5 U). DMS O (Dimethyl sulfoxide) was added to the reaction solution to a final concentration of 10% as needed.
- Each sample was amplified by DNA Engine PTC-0200 (MJ Research) or GeneAmpPCR System 9700 (PE Applied Biosystem).
- the PCR reaction was 94 after 2 minutes at 95 ° C. C 30 seconds, 58 ° C or 60. After 37 cycles of C 30 seconds and 72 ° C. for 3 minutes, the final extension reaction was performed at 72 ° C. for 10 minutes.
- each PCR product contains the polymorphism identified above for all genomic DNA samples.
- the reaction was performed in the same manner as described above using each primer set, and the analysis was performed.
- Tables 1 to 6 show the six polymorphisms that were strongly associated with granulocytopenia induced by taking vesnarinone as a result of this analysis, and their statistical values.
- the polymorphism indicated by “del” indicates a deletion polymorphism
- the number indicated by “polymorphism” indicates the ATG of the translation initiation codon ATG of the coding region of the IRS-2 gene. It is counted as + 1.
- those with minus (-1) in the numbers indicate that they are located 5 'upstream from A of the translation initiation codon ATG of the coding region of the IRS-2 gene.
- Table 7 shows the results of examining whether linkage disequilibrium is established between these polymorphisms.
- Example 3 This example relates to another method for detecting six polymorphisms of the human IRS-2 gene of the present invention, and was carried out as shown in the following (a) and (b).
- each DNA fragment was amplified by DNA Engine PTC-0200 (manufactured by MJ Research) or GeneAmpPCR System 9700 (manufactured by PE Applied Biosystems).
- Each PCR reaction was carried out at 95 ° C for 2 minutes, at 94 ° C for 30 seconds, at each annealing temperature shown in Table 8, 30 seconds, and at 72 ° C, for each elongation reaction time shown in Table 8, for 37 cycles.
- a final extension reaction was performed at 10 ° C. for 10 minutes.
- the annealing temperature and elongation reaction time are as shown in Table 8 below, for each DNA fragment, a temperature of 58 ° C to 60 ° C and a time of 0.5 minutes to 3 minutes, respectively.
- Example 2- The composition of the reaction solution is as shown in Example 2- (a). However, DMSO was added to the reaction solution for detecting “A-1164C” to a final concentration of 10% (DMSO in Table 8). Section).
- the 23rd G of the reverse primer (SEQ ID NO: 14) used for the detection of “A29793G” is a mutation inserted to introduce a restriction enzyme Afa I recognition site.
- nucleotide direct sequencing method (Dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)] or Polymorphism was detected by the Maxam-Gilbert method (Methods in Enzymology, 65, 499 (1980)), and the primers used are shown in Table 9 (SEQ ID NOs: 3, 6, 9, 12, and 17).
- A29793G was detected by PCR-RFLP (restriction fragment length polymorphism) analysis. That is, a 20 / iL reaction solution was prepared from 10 / L PCR product, 2 units of restriction enzyme Afa I (10un its / mL; manufactured by Takara) and 2 ⁇ L of 10X Buffer T attached to the restriction enzyme. To this, BSA was added to a final concentration of 0.01%, and after incubation at 37 ° C for 16 hours, the restriction enzyme-treated product was separated on a 4% agarose gel.
- the present invention has already reported the side effects of drug-induced granulocytopenia (including agranulocytosis) in the examination and determination of the risk of developing human drug-induced granulocytopenia, particularly by drug administration. Useful for examining and determining the risk of developing granulocytopenia before administration of the drug
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2530168A CA2530168C (en) | 2003-07-29 | 2004-07-28 | Method of judging risk for drug-induced granulocytopenia |
ES04770985T ES2379811T3 (es) | 2003-07-29 | 2004-07-28 | Procedimiento para juzgar el riesgo de granulocitopenia inducida por fármacos |
AT04770985T ATE544852T1 (de) | 2003-07-29 | 2004-07-28 | Verfahren zur beurteilung der gefahr arzneimittelinduzierter granulozytopenie |
KR1020127013518A KR101176191B1 (ko) | 2003-07-29 | 2004-07-28 | 약제 기인성 과립구 감소증 발증 리스크 판정법 |
US10/563,818 US7790372B2 (en) | 2003-07-29 | 2004-07-28 | Method of judging risk for onset of drug-induced granulocytopenia |
EP04770985A EP1650300B1 (en) | 2003-07-29 | 2004-07-28 | Method of judging risk for drug-induced granulocytopenia |
AU2004259952A AU2004259952B2 (en) | 2003-07-29 | 2004-07-28 | Method of judging risk for onset of drug-induced granulocytopenia |
KR1020057024365A KR101176194B1 (ko) | 2003-07-29 | 2005-12-19 | 약제 기인성 과립구 감소증 발증 리스크 판정법 |
US12/833,466 US20100273175A1 (en) | 2003-07-29 | 2010-07-09 | Method of judging risk for onset of drug-induced granulocytopenia |
US12/833,611 US8492087B2 (en) | 2003-07-29 | 2010-07-09 | Method of judging risk for onset of drug-induced granulocytopenia |
US13/917,041 US20140186826A1 (en) | 2003-07-29 | 2013-06-13 | Method of judging risk for onset of drug-induced granulocytopenia |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003281937 | 2003-07-29 | ||
JP2003-281937 | 2003-07-29 |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/563,818 A-371-Of-International US7790372B2 (en) | 2003-07-29 | 2004-07-28 | Method of judging risk for onset of drug-induced granulocytopenia |
US12/833,611 Division US8492087B2 (en) | 2003-07-29 | 2010-07-09 | Method of judging risk for onset of drug-induced granulocytopenia |
US12/833,466 Division US20100273175A1 (en) | 2003-07-29 | 2010-07-09 | Method of judging risk for onset of drug-induced granulocytopenia |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005010183A1 true WO2005010183A1 (ja) | 2005-02-03 |
Family
ID=34100975
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/010722 WO2005010183A1 (ja) | 2003-07-29 | 2004-07-28 | 薬剤起因性顆粒球減少症発症リスク判定法 |
Country Status (10)
Country | Link |
---|---|
US (4) | US7790372B2 (ja) |
EP (2) | EP2275539B1 (ja) |
KR (2) | KR101176191B1 (ja) |
CN (1) | CN100482794C (ja) |
AT (1) | ATE544852T1 (ja) |
AU (1) | AU2004259952B2 (ja) |
CA (1) | CA2530168C (ja) |
ES (2) | ES2391076T3 (ja) |
TW (1) | TWI351436B (ja) |
WO (1) | WO2005010183A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015136347A (ja) * | 2014-01-24 | 2015-07-30 | 大塚製薬株式会社 | 化合物の無顆粒球症誘発性の評価方法 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009062152A1 (en) * | 2007-11-09 | 2009-05-14 | Washington University In St. Louis | Methods for measuring the metabolism of cns derived biomolecules in vivo |
WO2010011506A2 (en) * | 2008-07-23 | 2010-01-28 | The Washington University | Risk factors and a therapeutic target for neurodegenerative disorders |
CN102791862B (zh) * | 2009-12-31 | 2017-04-05 | 库尔纳公司 | 通过抑制胰岛素受体底物2(irs2)和转录因子e3(tfe3)的天然反义转录物而治疗irs2相关疾病 |
CA2962969C (en) | 2014-09-30 | 2023-03-21 | Washington University | Tau kinetic measurements |
JP2022525742A (ja) | 2019-03-14 | 2022-05-19 | テルモ ビーシーティー バイオテクノロジーズ,エルエルシー | 凍結乾燥用ローディングトレイ組立体及びシステム |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4683202A (en) * | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
CA2020958C (en) | 1989-07-11 | 2005-01-11 | Daniel L. Kacian | Nucleic acid sequence amplification methods |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US6582908B2 (en) * | 1990-12-06 | 2003-06-24 | Affymetrix, Inc. | Oligonucleotides |
US5474796A (en) * | 1991-09-04 | 1995-12-12 | Protogene Laboratories, Inc. | Method and apparatus for conducting an array of chemical reactions on a support surface |
US5732076A (en) * | 1995-10-26 | 1998-03-24 | Omnipoint Corporation | Coexisting communication systems |
JP4362150B2 (ja) | 1996-11-29 | 2009-11-11 | サード ウェーブ テクノロジーズ,インコーポレーテッド | Fen−1エンドヌクレアーゼ、混合物、および開裂方法 |
US6159693A (en) | 1998-03-13 | 2000-12-12 | Promega Corporation | Nucleic acid detection |
JP2000279197A (ja) | 1999-03-31 | 2000-10-10 | Genome Science Laboratories Co Ltd | Hivの変異の検出方法 |
US20030092019A1 (en) * | 2001-01-09 | 2003-05-15 | Millennium Pharmaceuticals, Inc. | Methods and compositions for diagnosing and treating neuropsychiatric disorders such as schizophrenia |
US20030040315A1 (en) * | 2001-08-20 | 2003-02-27 | Farideh Khaleghi | Reduced state transition delay and signaling overhead for mobile station state transitions |
US8958493B2 (en) * | 2004-03-31 | 2015-02-17 | Infineon Technologies Ag | Operation for backward-compatible transmission |
US7764981B2 (en) * | 2004-07-30 | 2010-07-27 | Nokia Corporation | System and method for managing a wireless connection to reduce power consumption of a mobile terminal |
-
2004
- 2004-07-28 KR KR1020127013518A patent/KR101176191B1/ko not_active IP Right Cessation
- 2004-07-28 CN CNB2004800224990A patent/CN100482794C/zh not_active Expired - Fee Related
- 2004-07-28 CA CA2530168A patent/CA2530168C/en not_active Expired - Fee Related
- 2004-07-28 EP EP10010352A patent/EP2275539B1/en not_active Not-in-force
- 2004-07-28 WO PCT/JP2004/010722 patent/WO2005010183A1/ja active Application Filing
- 2004-07-28 AT AT04770985T patent/ATE544852T1/de active
- 2004-07-28 US US10/563,818 patent/US7790372B2/en not_active Expired - Fee Related
- 2004-07-28 ES ES10010352T patent/ES2391076T3/es active Active
- 2004-07-28 AU AU2004259952A patent/AU2004259952B2/en not_active Ceased
- 2004-07-28 ES ES04770985T patent/ES2379811T3/es active Active
- 2004-07-28 EP EP04770985A patent/EP1650300B1/en not_active Not-in-force
- 2004-07-29 TW TW093122778A patent/TWI351436B/zh not_active IP Right Cessation
-
2005
- 2005-12-19 KR KR1020057024365A patent/KR101176194B1/ko not_active IP Right Cessation
-
2010
- 2010-07-09 US US12/833,466 patent/US20100273175A1/en not_active Abandoned
- 2010-07-09 US US12/833,611 patent/US8492087B2/en not_active Expired - Fee Related
-
2013
- 2013-06-13 US US13/917,041 patent/US20140186826A1/en not_active Abandoned
Non-Patent Citations (5)
Title |
---|
BERNAL ET AL., DIABETES, vol. 47, 1998, pages 976 - 979 |
BROOKES, A.J.: "The essence of SNPs", GENE, vol. 234, 1999, pages 177 - 186, XP004173090 * |
MARTIN, E.R. ET AL.: "SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease", AM.J.HUM.GENET., vol. 67, 2000, pages 383 - 394, XP000995224 * |
MURAKAMI, Y. ET AL.: "Bioinformatics no Jissai", 2003, KODANSHA LTD., pages: 210 - 211, XP002985685 * |
SCHACHER, D.H. ET AL.: "Developmental expression of insulin receptor substrate-2 during dimethylsulfoxide-induced differentiation of human HL-60 cells", J.IMMUNOL., vol. 164, 2000, pages 113 - 120, XP002985686 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015136347A (ja) * | 2014-01-24 | 2015-07-30 | 大塚製薬株式会社 | 化合物の無顆粒球症誘発性の評価方法 |
Also Published As
Publication number | Publication date |
---|---|
EP1650300A1 (en) | 2006-04-26 |
US20100273176A1 (en) | 2010-10-28 |
AU2004259952A1 (en) | 2005-02-03 |
CN1833023A (zh) | 2006-09-13 |
US7790372B2 (en) | 2010-09-07 |
TW200516152A (en) | 2005-05-16 |
EP1650300A4 (en) | 2007-08-22 |
KR101176191B1 (ko) | 2012-08-22 |
ES2379811T3 (es) | 2012-05-03 |
US20140186826A1 (en) | 2014-07-03 |
EP2275539A3 (en) | 2011-05-18 |
CN100482794C (zh) | 2009-04-29 |
KR101176194B1 (ko) | 2012-08-22 |
EP1650300B1 (en) | 2012-02-08 |
EP2275539B1 (en) | 2012-09-05 |
TWI351436B (en) | 2011-11-01 |
US20070264631A1 (en) | 2007-11-15 |
KR20120068995A (ko) | 2012-06-27 |
CA2530168C (en) | 2014-05-27 |
AU2004259952B2 (en) | 2008-10-16 |
US8492087B2 (en) | 2013-07-23 |
CA2530168A1 (en) | 2005-02-03 |
ES2391076T3 (es) | 2012-11-21 |
ATE544852T1 (de) | 2012-02-15 |
US20100273175A1 (en) | 2010-10-28 |
KR20060040600A (ko) | 2006-05-10 |
EP2275539A2 (en) | 2011-01-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20140186826A1 (en) | Method of judging risk for onset of drug-induced granulocytopenia | |
US20080020383A1 (en) | Haplotype Markers And Methods Of Using The Same To Determine Response To Treatment | |
JP5078358B2 (ja) | 腎細胞癌に対するインターフェロン治療反応性識別マーカー | |
KR101141185B1 (ko) | 치료의 제안된 효능 검출용 마커 | |
JP4632712B2 (ja) | 薬剤起因性顆粒球減少症発症リスク判定法 | |
JP2006288279A (ja) | 出血時間延長傾向判定方法 | |
JP2002238577A (ja) | 脳動脈瘤感受性遺伝子 | |
US7618779B2 (en) | Chromosome 1p36 polymorphisms and low bone mineral density | |
JP2005027595A (ja) | 気管支喘息発症危険因子の検出方法 | |
WO2002059305A1 (fr) | Procede de detection d'un facteur etiologique d'asthme |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200480022499.0 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2004770985 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2530168 Country of ref document: CA Ref document number: 1020057024365 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10563818 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004259952 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12006500225 Country of ref document: PH |
|
ENP | Entry into the national phase |
Ref document number: 2004259952 Country of ref document: AU Date of ref document: 20040728 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2004259952 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2004770985 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1020057024365 Country of ref document: KR |
|
WWP | Wipo information: published in national office |
Ref document number: 10563818 Country of ref document: US |