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WO2011118968A2 - Polymorphisme mononucléotidique pour diagnostiquer la récurrence d'un carcinome hépatocellulaire - Google Patents

Polymorphisme mononucléotidique pour diagnostiquer la récurrence d'un carcinome hépatocellulaire Download PDF

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WO2011118968A2
WO2011118968A2 PCT/KR2011/001965 KR2011001965W WO2011118968A2 WO 2011118968 A2 WO2011118968 A2 WO 2011118968A2 KR 2011001965 W KR2011001965 W KR 2011001965W WO 2011118968 A2 WO2011118968 A2 WO 2011118968A2
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primer
igf2 gene
region
seq
amplifying
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WO2011118968A3 (fr
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정영화
유은실
이영주
김정아
이종은
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University of Ulsan Foundation for Industry Cooperation
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University of Ulsan Foundation for Industry Cooperation
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Priority to US13/636,806 priority Critical patent/US20130023442A1/en
Priority to CN201180025453.4A priority patent/CN102906276B/zh
Priority claimed from KR1020110025175A external-priority patent/KR101359782B1/ko
Publication of WO2011118968A2 publication Critical patent/WO2011118968A2/fr
Publication of WO2011118968A3 publication Critical patent/WO2011118968A3/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 relates to a single base polymorphism (SNP) useful for predicting recurrence after hepatocellular carcinoma surgery, a microarray for diagnosing recurrence of hepatocellular carcinoma or a diagnostic kit using the same, and a method for screening drugs for improving recurrence of hepatocellular carcinoma.
  • SNP single base polymorphism
  • Hepatocellular carcinoma is a very common and important cancer that occupies the third place among all malignant tumors in Korean cancer occurrence and death. Hepatocellular carcinoma is the most representative hypervascular tumor among malignant tumors.
  • MTA1 metastatic tumor antigen 1
  • HIF1 hypoxia inducible factor 1
  • VEGF vascular endothelial growth factor
  • SNPs single nucleotide polymorphisms
  • the present inventors have completed the present invention by finding a SNP that has a meaningful correlation with postoperative recurrence of hepatocellular carcinoma.
  • SNP single base polymorphism
  • SNP single nucleotide polymorphism
  • Another object of the present invention is to provide a kit for diagnosing recurrence of hepatocellular carcinoma.
  • Another object of the present invention is to provide a method for diagnosing recurrence of hepatocellular carcinoma using the single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • Another object of the present invention is to provide a method for screening a drug for preventing recurrence of hepatocellular carcinoma using the single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the present invention provides a C allele (CC or GC genotype) of -291C / G (rs3213221) [SEQ ID NO: 1] of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) [SEQ ID NO: 2] of the IGF2 gene; The T allele (CT or TT genotype) of 66378C / T (rs1048201) [SEQ ID NO: 3] of the FGF2 gene; G allele (GG or GA genotype) of 50012A / G (rs6534367) [SEQ ID NO: 4] of the FGF2 gene; GC haplotype of 6310 (rs2585) [SEQ ID NO: 5] / 4702 (rs3802971) [SEQ ID NO: 6] of the IGF2 gene; Homozygous CC haplotype of -11228 (rs2239681) [SEQ ID NO:
  • the recurrence of hepatocellular carcinoma may be related to recurrence after surgery in a patient who underwent radical resection for hepatocellular carcinoma.
  • the present invention also provides a microarray for recurrence diagnosis of hepatocellular carcinoma, characterized in that it comprises a polynucleotide of the single nucleotide polymorphism (SNP) for predicting recurrence of hepatocellular carcinoma, a polypeptide encoded therein or cDNA thereof.
  • SNP single nucleotide polymorphism
  • the present invention also provides a kit for diagnosing recurrence of hepatocellular carcinoma including the microarray.
  • the diagnostic kit according to the present invention may further include a primer set used to separate and amplify DNA containing the SNP from the subject in addition to the microarray of the present invention.
  • an embodiment of the present invention provides a kit for diagnosing recurrence of hepatocellular carcinoma using a single-base extension (SBE) reaction to genotype SNP.
  • SBE single-base extension
  • the kit for diagnosing recurrence of hepatocellular carcinoma using the single base extension reaction includes C allele (CC or CG genotype) of -291C / G (rs3213221) [SEQ ID NO: 1] of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) [SEQ ID NO: 2] of the IGF2 gene; Homozygous GC haplotype of 6310 (rs2585) [SEQ ID NO: 5] / 4702 (rs3802971) [SEQ ID NO: 6] of the IGF2 gene; And CT haplotype of -11228 (rs2239681) [SEQ ID NO: 7] /-13021 (rs3741208) [SEQ ID NO: 2] of the IGF2 gene.
  • the forward primer for amplifying the -13021 (rs3741208) region of the IGF2 gene Reverse primers for amplifying the -13021 (rs3741208) region of the IGF2 gene; Primers for genotyping the -13021 (rs3741208) region of the IGF2 gene; Forward primers for amplifying the 6310 (rs2585) region of the IGF2 gene; Reverse primers for amplifying the 6310 (rs2585) region of the IGF2 gene; Genotyping primer for amplifying the 6310 (rs2585) region of the IGF2 gene; Forward primers for amplifying the -11228 (rs2239681) region of the IGF2 gene; Reverse primers for amplifying the -11228 (rs2239681) region of the IGF2 gene; Primers for genotyping of the -11228 (rs2239681) region of the IGF2 gene; Forward primers for amplifying the 4702 (rs38029
  • the forward primer for amplifying the -13021 (rs3741208) region of the IGF2 gene in the kit for diagnosing recurrence of hepatocellular carcinoma includes a primer of SEQ ID NO: 17;
  • Reverse primers for amplifying the -13021 (rs3741208) region of the IGF2 gene include a primer of SEQ ID NO: 18;
  • the primer for genotyping of the -13021 (rs3741208) region of the IGF2 gene may include a primer of SEQ ID NO: 35;
  • Forward primer for amplifying the 6310 (rs2585) region of the IGF2 gene is a primer of SEQ ID NO: 20;
  • Reverse primer for amplifying the 6310 (rs2585) region of the IGF2 gene is a primer of SEQ ID NO: 21;
  • the primer for genotyping for amplifying the 6310 (rs2585) region of the IGF2 gene may include a primer of SEQ ID NO: 36;
  • the present invention comprises the steps of obtaining a nucleic acid sample from the sample; And the C allele (CC or GC genotype) of -291C / G (rs3213221) of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) of the IGF2 gene; T allele (CT or TT genotype) of 66378C / T (rs1048201) of the FGF2 gene; G allele (GG or GA genotype) of 50012A / G (rs6534367) of the FGF2 gene; GC haplotype of 6310 (rs2585) / 4702 (rs3802971) of the IGF2 gene; Homozygous CC haplotype of 11228 (rs2239681) /-13021 (rs3741208) of the IGF2 gene; And determining the nucleotide sequence of any one or more polymorphic sites of at least one polynu
  • Determining the nucleotide sequence of the polymorphic site may comprise hybridizing the nucleic acid sample to a microarray to which the polynucleotide or its complementary nucleotide is immobilized and detecting the obtained hybridization result.
  • the present invention comprises the steps of contacting the candidate with a polypeptide encoded by the polynucleotide of the single nucleotide polymorphism (SNP) or a complementary nucleotide thereof for the diagnosis of recurrence of hepatocellular carcinoma; And it provides a method for screening a drug for preventing recurrence of hepatocellular carcinoma characterized in that it comprises the step of determining whether the candidate exhibits the activity to enhance the function or inhibit the function of the polypeptide.
  • SNP single nucleotide polymorphism
  • SNP single base polymorphism
  • Figure 1 shows the cumulative recurrence rate of hepatocellular carcinoma according to genotype at the -291C / G (rs3213221) position of the IGF2 gene
  • Figure 2 shows the cumulative recurrence rate of hepatocellular carcinoma according to genotype at -13021C / T (rs3741208) position of the IGF2 gene
  • Figure 3 shows the cumulative recurrence rate of hepatocellular carcinoma according to the GC haplotype of 6310 (rs2585) / 4702 (rs3802971) of the IGF2 gene
  • Figure 4 shows the cumulative recurrence rate of hepatocellular carcinoma according to the CC haplotype of -11228 (rs2239681) / -13021 (rs3741208) of the IGF2 gene
  • Figure 5 shows the cumulative recurrence rate of hepatocellular carcinoma according to CT haplotype of -11228 (rs2239681) / -13021 (rs3741208) of the IGF2 gene
  • 6 to 8 show genotyping results for confirming whether a single base polymorphism is used for diagnosing recurrence of hepatocellular carcinoma using a kit for diagnosing recurrence of hepatocellular carcinoma according to an embodiment of the present invention.
  • the present invention provides a C allele (CC or GC genotype) of -291C / G (rs3213221) [SEQ ID NO: 1] of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) [SEQ ID NO: 2] of the IGF2 gene; The T allele (CT or TT genotype) of 66378C / T (rs1048201) [SEQ ID NO: 3] of the FGF2 gene; G allele (GG or GA genotype) of 50012A / G (rs6534367) [SEQ ID NO: 4] of the FGF2 gene; GC haplotype of 6310 (rs2585) [SEQ ID NO: 5] / 4702 (rs3802971) [SEQ ID NO: 6] of the IGF2 gene; Homozygous CC haplotype of -11228 (rs2239681) [SEQ ID NO:
  • the recurrence of hepatocellular carcinoma may be related to recurrence after surgery in a patient who underwent radical resection for hepatocellular carcinoma.
  • the present invention also provides a microarray for recurrence diagnosis of hepatocellular carcinoma, characterized in that it comprises a polynucleotide of the single nucleotide polymorphism (SNP) for predicting recurrence of hepatocellular carcinoma, a polypeptide encoded therein or cDNA thereof.
  • SNP single nucleotide polymorphism
  • the microarray for diagnosing recurrence of hepatocellular carcinoma can be prepared by conventional methods known to those skilled in the art.
  • the polynucleotides constituting the microarray for diagnosing recurrence of hepatocellular carcinoma include amino-silane, poly-L-lysine, and
  • the active group selected from the group consisting of aldehydes can be fixed to the coated substrate, and the substrate can be selected from the group consisting of silicon wafers, glass, quartz, metals and plastics.
  • a method of immobilizing the polynucleotide on a substrate a micropipetting method using a piezoelectric method, a method using a pin type spotter, or the like can be used.
  • the present invention also provides a kit for diagnosing recurrence of hepatocellular carcinoma including the microarray.
  • the diagnostic kit according to the present invention may further include a primer set used to separate and amplify DNA containing the SNP from the subject in addition to the microarray of the present invention.
  • a primer set used to separate and amplify DNA containing the SNP from the subject in addition to the microarray of the present invention.
  • an embodiment of the present invention provides a kit for diagnosing recurrence of hepatocellular carcinoma using a single-base extension (SBE) reaction to genotype SNP.
  • SBE single-base extension
  • amplification (forward and reverse) and extension (genotyping) primers should be designed for single-base extension (SBE).
  • Kit for diagnosing recurrence of hepatocellular carcinoma using the single base extension reaction may be a kit for genotyping of the SNaPshot method.
  • the kit for diagnosing recurrence of hepatocellular carcinoma using the single base extension reaction includes C allele (CC or CG genotype) of -291C / G (rs3213221) [SEQ ID NO: 1] of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) [SEQ ID NO: 2] of the IGF2 gene; Homozygous GC haplotype of 6310 (rs2585) [SEQ ID NO: 5] / 4702 (rs3802971) [SEQ ID NO: 6] of the IGF2 gene; And CT haplotype of -11228 (rs2239681) [SEQ ID NO: 7] /-13021 (rs3741208) [SEQ ID NO: 2] of the IGF2 gene.
  • the forward primer for amplifying the -13021 (rs3741208) region of the IGF2 gene Reverse primers for amplifying the -13021 (rs3741208) region of the IGF2 gene; Primers for genotyping the -13021 (rs3741208) region of the IGF2 gene; Forward primers for amplifying the 6310 (rs2585) region of the IGF2 gene; Reverse primers for amplifying the 6310 (rs2585) region of the IGF2 gene; Genotyping primer for amplifying the 6310 (rs2585) region of the IGF2 gene; Forward primers for amplifying the -11228 (rs2239681) region of the IGF2 gene; Reverse primers for amplifying the -11228 (rs2239681) region of the IGF2 gene; Primers for genotyping of the -11228 (rs2239681) region of the IGF2 gene; Forward primers for amplifying the 4702 (rs38029
  • the forward primer for amplifying the -13021 (rs3741208) region of the IGF2 gene in the kit for diagnosing recurrence of hepatocellular carcinoma includes a primer of SEQ ID NO: 17;
  • Reverse primers for amplifying the -13021 (rs3741208) region of the IGF2 gene include a primer of SEQ ID NO: 18;
  • the primer for genotyping of the -13021 (rs3741208) region of the IGF2 gene may include a primer of SEQ ID NO: 35;
  • Forward primer for amplifying the 6310 (rs2585) region of the IGF2 gene is a primer of SEQ ID NO: 20;
  • Reverse primer for amplifying the 6310 (rs2585) region of the IGF2 gene is a primer of SEQ ID NO: 21;
  • the primer for genotyping for amplifying the 6310 (rs2585) region of the IGF2 gene may include a primer of SEQ ID NO: 36;
  • the present invention comprises the steps of obtaining a nucleic acid sample from the sample; And the C allele (CC or GC genotype) of -291C / G (rs3213221) of the IGF2 gene; T allele (CT or TT genotype) of -13021C / T (rs3741208) of the IGF2 gene; T allele (CT or TT genotype) of 66378C / T (rs1048201) of the FGF2 gene; G allele (GG or GA genotype) of 50012A / G (rs6534367) of the FGF2 gene; GC haplotype of 6310 (rs2585) / 4702 (rs3802971) of the IGF2 gene; Homozygous CC haplotype of 11228 (rs2239681) /-13021 (rs3741208) of the IGF2 gene; And determining the nucleotide sequence of any one or more polymorphic sites of at least one polynu
  • the nucleic acid may comprise cDNA synthesized from DNA, mRNA or mRNA.
  • Determining the nucleotide sequence of the polymorphic site may comprise hybridizing the nucleic acid sample to a microarray to which the polynucleotide or its complementary nucleotide is immobilized and detecting the obtained hybridization result.
  • DNA is isolated from tissues, body fluids, or cells of a subject, and then amplified by PCR, followed by SNP analysis.
  • SNP analysis can be performed by conventional methods known in the art.
  • the SNP analysis may be performed using a real-time PCR system, or may be performed through determination of the nucleotide sequence of the nucleic acid directly by the dideoxy method, or a probe including the sequence of the SNP site or a probe complementary thereto.
  • the present invention comprises the steps of contacting the candidate with a polypeptide encoded by the polynucleotide of the single nucleotide polymorphism (SNP) or a complementary nucleotide thereof for the diagnosis of recurrence of hepatocellular carcinoma; And it provides a method for screening a drug for preventing recurrence of hepatocellular carcinoma characterized in that it comprises the step of determining whether the candidate exhibits the activity to enhance the function or inhibit the function of the polypeptide.
  • SNP single nucleotide polymorphism
  • the reaction between the polypeptide and the candidate may be used in the conventional methods used to confirm the reaction between the protein-protein and the protein-compound.
  • a method for measuring activity after reacting the protein with a candidate substance a yeast two-hybrid method, a search for phage-displayed peptide clones that bind to the protein, and natural products
  • high throughput screening using a chemical library, drug hit HTS, cell-based screening, or screening using a DNA array can be used.
  • the candidate material may be individual nucleic acids, proteins, other extracts or natural products, compounds, or the like, which are estimated to have the potential as a diagnostic agent for recurrence of hepatocellular carcinoma according to a conventional selection method, or randomly selected. have.
  • Target SNPs ID is as follows.
  • haplotype frequency is at least 5% of the SNPs using PHASE software v2.1 analyzed the incidence of haplotypes, and Haploview program v3.2 (http: //www.broad Linkage disequilibrium was analyzed using .mit.edu / mpg / haploview / index.php ).
  • a primer set capable of amplifying a region including the SNP of Example 1 and a TaqMan probe including the SNP region were prepared using primer express software. TaqMan probes were prepared for each wild type and mutant allele according to the sequence of the SNP.
  • One side of the TaqMan probe tagged the fluorescent dye and the other side produced a probe by tagging a quencher capable of suppressing the color of the fluorescent dye.
  • the fluorescent dyes of different colors were tagged with the wild type and the mutant alleles.
  • the results of SNP marker analysis were judged as final results by verifying the agreement of two or more independent callers.
  • the SNP marker results of the individuals are shown as major allele homozygote, heterozygote, and minor allele homozygote according to single nucleotide polymorphic alleles. Results for all subjects were expressed by the ratio of major alleles to minor alleles and the frequency of three genotypes, and the Hardy-Weinberg equilibrium was verified.
  • CT or TT genotype 66378 C / T (rs1048201)
  • G allele GG or GA genotype
  • the cumulative recurrence rates at 1 year were 32.5% and 22%, respectively, as in Figure 1, and the cumulative recurrence rates at 2 and 3 years were 46.9% and 34.5%, respectively. With 64.4% ,. 39.6%. As shown in FIG.
  • the T allele (CT or TT genotype) at the -13021 C / T (rs3741208) position of the IGF2 gene showed a significantly higher postoperative cumulative recurrence rate than the CC genotype (CT or TT).
  • CT or TT genotype the T allele (CT or TT genotype) at the -13021 C / T (rs3741208) position of the IGF2 gene showed a significantly higher postoperative cumulative recurrence rate than the CC genotype (CT or TT).
  • the CC haplotype and CT haplotype showed significant correlation at the -11228 (rs2239681) /-13021 (rs3741208) position of the IGF2 gene.
  • SEQ ID NO: 35 For SEQ ID NO: 35 (5′-GCCTSCTGACCACCAGCAAGAAATTGGACAGGAGACYGARGAGAAA-3 ′), SEQ ID NO: 46 (5′-GCCTGCTGACCACCAGCAAGAAATTGGACAGGAGACCGAAGAGAAA-3 ′), SEQ ID NO: 47 (5′-GCCTGCTGACCACCAGCAAGAAATTAGACAGGAGAGAGGAGAGAGAGGAGGAGAGGAGA) -3 '), SEQ ID NO: 49 (5'-GCCTCCTGACCACCAGCAAGAAATTGGACAGGAGACTGAAGAGAAA-3'), SEQ ID NO: 50 (5'-GCCTGCTGACCACCAGCAAGAAATTGGACAGGAGACCGAGGAGAAA-3 '), SEQ ID NO: 51 (5'-GCCTGCTGACCACCACCAGGAAGAA'GAACAGGA'GAGA) -GCCTCCTGACCACCAGCAAGAAATTGGACAGGAGACCGAGGAAA-3 ') and SEQ ID NO: 53 (5'-GCCTCCTG
  • Sites containing single nucleotide polymorphism were first amplified by multiplex PCR.
  • the composition and PCR reaction conditions of the PCR reaction solution used in the PCR reaction are shown in Tables 3 and 4, respectively. More specifically, DNA was isolated from the sample sample and used as template DNA for the PCR reaction.
  • the PCR reaction was predenatured at about 95 ° C. for about 15 minutes (1 cycle), denaturation at about 94 ° C. for about 30 seconds, annealing at about 55 ° C. for about 1 minute and 30 seconds, and about 72 ° C.
  • the condition of elongation at about 1 minute 30 seconds was final elongation at 35 cycles of 1 cycle at about 72 ° C. for about 10 minutes.
  • the reaction was finally held at about 4 ° C.
  • forward and reverse primers for each single base polymorphism of Table 2 were used as primers for the PCR reaction.
  • the purified PCR product was mixed with SNaPshot Reaction Premix (Applied Biosystems, CA, USA) and genotyping primers for each single base polymorphism of Table 2, followed by PCR reaction.
  • the composition and reaction conditions of the SNaPshot reactant are shown in Tables 8 and 9, respectively.
  • the PCR reaction was carried out for 25 cycles of 1 cycle of denaturation at about 96 ° C., annealing at about 50 ° C., annealing at about 50 ° C., and elongation at about 60 ° C. for about 30 seconds.
  • SAP was added to the SNaPshot reaction product.
  • the composition and reaction conditions of the SAP treated reactants are shown in Tables 9 and 10, respectively.
  • ABI 3730XL which is an automatic sequencing device
  • ABI 3730XL which is an automatic sequencing device
  • only a labeled portion is detected and shown as a peak as shown in FIGS. 6 to 8.
  • the fluorescence color represented by the peak was analyzed (using ddNTP labeled with a fluorescent dye of different color for each base) to confirm the sequence of the single nucleotide polymorphism site.
  • a single base polymorphism site useful for predicting recurrence after hepatocellular carcinoma surgery Although single nucleotide polymorphisms of rs3741208, rs2585, rs2239681, and rs3213221 could be analyzed in multiples, rs3802971, a single nucleotide polymorphism site useful for predicting recurrence after hepatocellular carcinoma surgery, could not be multiplexed and a single analysis was needed. appear.
  • SNP single base polymorphism
  • SEQ ID NO: 1 shows the sequence of -291C / G (rs3213221) of the human IGF2 gene
  • SEQ ID NO: 2 shows the sequence of -13021C / T of the human IGF2 gene
  • SEQ ID NO: 3 shows the sequence of 66378C / T (rs1048201) of the human FGF2 gene
  • SEQ ID NO: 4 shows the sequence of 50012A / G (rs6534367) of the human FGF2 gene
  • SEQ ID NO: 5 shows the sequence of 6310 (rs2585) of the human IGF2 gene
  • SEQ ID NO: 6 shows the sequence of 4702 (rs3802971) of the human IGF2 gene
  • SEQ ID NO: 7 shows the sequence of -11228 (rs2239681) of the human IGF2 gene
  • SEQ ID NO: 8 shows the sequence of the forward primer of rs1048201
  • SEQ ID NO: 9 shows the sequence of a reverse primer of rs1048201
  • SEQ ID NO: 10 shows a sequence of a genotyping primer of rs1048201
  • SEQ ID NO: 11 shows the sequence of a forward primer of rs6534367
  • SEQ ID NO: 12 shows the sequence of a reverse primer of rs6534367
  • SEQ ID NO: 13 shows a sequence of a genotyping primer of rs6534367
  • SEQ ID NO: 14 shows the sequence of a forward primer of rs308428
  • SEQ ID NO: 15 shows the sequence of a reverse primer of rs308428
  • SEQ ID NO: 16 shows the sequence of a genotyping primer of rs308428
  • SEQ ID NO: 17 shows the sequence of a forward primer of rs3741208
  • SEQ ID NO: 18 shows the sequence of a reverse primer of rs3741208
  • SEQ ID NO: 19 shows a sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 20 shows the sequence of a forward primer of rs2585
  • SEQ ID NO: 21 shows the sequence of a reverse primer of rs2585
  • SEQ ID NO: 22 shows a sequence of a genotyping primer of rs2585
  • SEQ ID NO: 23 shows the sequence of a forward primer of DL1002505,
  • SEQ ID NO: 24 shows the sequence of a reverse primer of DL1002505,
  • SEQ ID NO: 25 shows the sequence of a genotyping primer of DL1002505,
  • SEQ ID NO: 26 shows the sequence of a forward primer of rs308395
  • SEQ ID NO: 27 shows the sequence of a reverse primer of rs308395
  • SEQ ID NO: 28 shows the sequence of a genotyping primer of rs308395
  • SEQ ID NO: 29 shows the sequence of a forward primer of rs11938826,
  • SEQ ID NO: 30 shows the sequence of a reverse primer of rs11938826,
  • SEQ ID NO: 31 shows a sequence of a genotyping primer of rs11938826,
  • SEQ ID NO: 32 shows the sequence of a forward primer of rs3213221
  • SEQ ID NO: 33 shows the sequence of a reverse primer of rs3213221
  • SEQ ID NO: 34 shows the sequence of a genotyping primer of rs3213221
  • SEQ ID NO: 35 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 36 shows the sequence of a genotyping primer of rs2585
  • SEQ ID NO: 37 shows the sequence of a forward primer of rs2239681,
  • SEQ ID NO: 38 shows the sequence of a reverse primer of rs2239681,
  • SEQ ID NO: 39 shows the sequence of a genotyping primer of rs2239681,
  • SEQ ID NO: 40 shows the sequence of a forward primer of rs3802971
  • SEQ ID NO: 41 shows the sequence of a reverse primer of rs3802971
  • SEQ ID NO: 42 shows a sequence of a genotyping primer of rs3802971
  • SEQ ID NO: 43 shows the sequence of a forward primer of rs3213221
  • SEQ ID NO: 44 shows the sequence of a reverse primer of rs3213221
  • SEQ ID NO: 45 shows a sequence of a genotyping primer of rs3213221
  • SEQ ID NO: 46 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 47 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 48 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 49 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 50 shows the sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 51 shows a sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 52 shows a sequence of a genotyping primer of rs3741208
  • SEQ ID NO: 53 shows the sequence of a genotyping primer of rs3741208.

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Abstract

L'invention concerne un polymorphisme mononucléotidique (SNP) servant à diagnostiquer la récurrence d'un carcinome hépatocellulaire, le SNP montrant une corrélation significative avec le risque de récurrence d'un carcinome hépatocellulaire après un traitement chirurgical du carcinome hépatocellulaire. Ainsi, le SNP peut être utilisé pour développer des puces à ADN ou des trousses de diagnostic pour diagnostiquer la récurrence d'un carcinome hépatocellulaire, et pour cribler des médicaments permettant d'éviter la récurrence d'un carcinome hépatocellulaire, ce qui permet d'éviter la récurrence d'un carcinome hépatocellulaire après un traitement chirurgical du carcinome hépatocellulaire.
PCT/KR2011/001965 2010-03-22 2011-03-22 Polymorphisme mononucléotidique pour diagnostiquer la récurrence d'un carcinome hépatocellulaire Ceased WO2011118968A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US13/636,806 US20130023442A1 (en) 2010-03-22 2011-03-22 Single nucleotide polymorphism for predicting recurrence of hepatocellular carcinoma
CN201180025453.4A CN102906276B (zh) 2010-03-22 2011-03-22 用于对肝细胞癌复发进行预测的单核苷酸多态性

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2010-0025372 2010-03-22
KR20100025372 2010-03-22
KR1020110025175A KR101359782B1 (ko) 2010-03-22 2011-03-22 간세포암종의 재발 진단용 단일 염기 다형성
KR10-2011-0025175 2011-03-22

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WO2011118968A2 true WO2011118968A2 (fr) 2011-09-29
WO2011118968A3 WO2011118968A3 (fr) 2012-03-08

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PCT/KR2011/001965 Ceased WO2011118968A2 (fr) 2010-03-22 2011-03-22 Polymorphisme mononucléotidique pour diagnostiquer la récurrence d'un carcinome hépatocellulaire

Country Status (1)

Country Link
WO (1) WO2011118968A2 (fr)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI 16 July 2005 Database accession no. rs3741208 *
DATABASE NCBI 17 October 2007 Database accession no. rs3802971 *
DATABASE NCBI 26 February 2008 Database accession no. rs2585 *
DATABASE NCBI 28 August 2007 Database accession no. rs2239681 *
KIM, Y. J. ET AL.: 'IGF2 polymorphisms are associated with hepatitis B virus clearance and hepatocellular carcinoma' BIOCHEM. BIOPHYS. RES. COMMUN. vol. 346, no. 1, 21 July 2006, pages 38 - 44 *
TAKEDA, S.: 'Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease' ONCOGENE vol. 12, no. 7, 04 April 1996, pages 1589 - 1592 *

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