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WO2017222247A2 - Marqueur de prédiction de l'efficacité d'un agent thérapeutique contre l'hémophilie et son utilisation - Google Patents

Marqueur de prédiction de l'efficacité d'un agent thérapeutique contre l'hémophilie et son utilisation Download PDF

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WO2017222247A2
WO2017222247A2 PCT/KR2017/006344 KR2017006344W WO2017222247A2 WO 2017222247 A2 WO2017222247 A2 WO 2017222247A2 KR 2017006344 W KR2017006344 W KR 2017006344W WO 2017222247 A2 WO2017222247 A2 WO 2017222247A2
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hemophilia
seq
base
efficacy
therapeutic agent
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Korean (ko)
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WO2017222247A3 (fr
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조은해
이태헌
전영주
황태주
유기영
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Korea Hemophilia Foundation
GC Genome Corp
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Korea Hemophilia Foundation
Green Cross Genome Corp
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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
    • 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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a marker for predicting the efficacy of a hemophilia therapeutic agent, and more particularly, a single nucleotide polymorphism (SNP) marker for predicting the efficacy of a hemophilia therapeutic agent, a composition comprising an agent capable of detecting the SNP marker, and a kit comprising the composition.
  • a microarray a method for providing information for predicting the efficacy of the hemophilia therapeutic agent using the SNP markers, and a method for selecting the SNP for predicting the efficacy of the hemophilia therapeutic agent.
  • Hemophilia is a hemorrhagic disease due to a lack of coagulation factor.
  • Factor VIII deficiency is referred to as hemophilia A and factor IX deficiency as hemophilia B.
  • the incidence of hemophilia A occurs in 5,000 to 1 person per 10,000, and the incidence of hemophilia B is estimated to be about 1/5 of that (Park YS. J Korean Med Assoc, 2009; 52 (12): 1201-). 6).
  • Hemophilia is classified as severe ( ⁇ 1%), moderate (1-5%), or mild (> 5%), depending on the activity of the coagulation factor.
  • hemophilia treatment is used to compensate for the lack of coagulation factors.
  • Factor VIII and Factor IX preparations are currently used, and bypasses are administered when antibodies are generated during treatment (Kemton CL et al., Blood. 2009; 113 (1). ): 11-7).
  • the frequency of antibody production response in hemophilia A patients is about 30% and in hemophilia B patients by about 3% (Kessler CM, Hematology Am Soc Hematol Educ Program. 2005: 429-35).
  • Antibodies to hemophilia drugs occur most frequently within 50 days after drug exposure and are typically classified as antibody-producing groups above 0.6 Bethesda Unit (BU), and above 5 BU into hyperantibody-producing groups (Kasper CK et al. Thromb Diath Haemorrh. 1975; 34 (2): 612; Verbruggen B et al., Thromb Haemost.
  • the inventors of the present application performed a full-length genome wide association study using exome sequencing data to identify genetic predictive markers of the antibody-producing response of hemophilia treatment agents. A set of related SNPs capable of predicting these was identified and the present invention was completed.
  • Another object of the present invention is to provide a composition for predicting the efficacy of a hemophilia therapeutic agent, including an agent capable of detecting the SNP marker for predicting the efficacy of the hemophilia therapeutic agent.
  • Still another object of the present invention is to provide a kit or microarray for predicting the efficacy of a hemophilia therapeutic agent comprising the composition for predicting the efficacy of the hemophilia therapeutic agent.
  • Still another object of the present invention is to provide a method of providing information for predicting the efficacy of a hemophilia therapeutic agent, comprising determining a base of a polymorphic site of the SNP marker.
  • Still another object of the present invention is to provide a method for selecting an SNP marker for predicting the efficacy of a hemophilia therapeutic agent.
  • the present invention is derived from the nucleotide sequence of SEQ ID NO: 1 to 87, five or more consecutive comprising a 101 base of SEQ ID NO: 1 to 84 and 100 base of SEQ ID NO: 85 to 87 as SNP
  • SNP Single Nucleotide Polymorphism
  • the present invention also provides a composition for predicting the efficacy of a hemophilia therapeutic agent, comprising a probe, a primer or an aptamer capable of detecting the SNP marker.
  • the present invention also provides a use of the composition for predicting the efficacy of a hemophilia therapeutic agent.
  • the present invention also provides a kit or microarray for predicting the efficacy of a hemophilia therapeutic agent comprising the composition.
  • the present invention also provides a method for predicting the efficacy of a hemophilia therapeutic agent using the composition.
  • the present invention also comprises the steps of (a) amplifying a polymorphic site of the SNP marker from the DNA of the isolated sample or hybridizing with the probe; And (b) determining the base of the amplified or hybridized polymorphic site of step (a), thereby providing information for predicting the efficacy of the hemophilia therapeutic agent.
  • the present invention also relates to (a) screening SNPs having a sequencing depth value of 10 or more and a genotype quality value of 20 or more in the whole length exome sequencing of a subject to which hemophilia treatment is administered. Removing an intergenic variant having no residue region therein; (b) performing an associative analysis on the SNPs selected in step (a) to select the SNPs associated with the hemophilia therapeutic antibody antibody phenotype; And (c) selecting the SNPs of the gene regions related to the immune system with respect to the SNPs selected in step (b).
  • FIG. 1 is a schematic diagram showing a method of selecting an SNP marker predicting efficacy of a hemophilia therapeutic agent.
  • the present invention provides a single nucleotide polymorphism (SNP) marker, which is a monobasic polymorphism predictive of a hemophilia therapeutic agent.
  • SNP single nucleotide polymorphism
  • the marker is preferably derived from the nucleotide sequences of SEQ ID NOs: 1 to 87, consisting of 5 or more consecutive bases comprising the 101 base of SEQ ID NOs: 1 to 84 and the 100 base of SEQ ID NOs: 85 to 87 as SNPs. It may be a Single Nucleotide Polymorphism (SNP) marker predicting the efficacy of a hemophilia therapeutic agent comprising one or more polynucleotides or complementary polynucleotides selected from polynucleotides.
  • SNP Single Nucleotide Polymorphism
  • hemophilia is a hemorrhagic disease due to coagulation factor deficiency
  • factor VIII deficiency is referred to as hemophilia A
  • factor IX deficiency is referred to as hemophilia B
  • Hemophilia A is a hemorrhagic disease characterized by quantitative or qualitative defects of FVIII due to defects in the FVIII gene
  • hemophilia B is characterized by quantitative or qualitative defects of FIX due to abnormalities in the FIX gene.
  • the present invention relates to the prediction of efficacy of hemophilia A therapeutics.
  • polymorphism refers to a case in which two or more alleles exist in one locus, and among polymorphism sites, a single base differs according to a person. It is called single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles that exhibit a frequency of occurrence of at least 1%, more preferably at least 5% or 10% in the selected population.
  • allele refers to several types of genes that exist at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphism, for example SNPs have two kinds of bialleles.
  • rs_id used in the present invention means rs-ID, which is an independent marker assigned to all SNPs initially registered by NCBI, which began accumulating SNP information since 1998. In the present invention, it is described in the form of rs2486316. Rs_id described in such a table means an SNP marker which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the position and sequence of the SNP using the rs_id. The specific sequence corresponding to NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number rs_id may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also applies to such altered sequences.
  • SEQ ID NOs 1 to 87 is a polymorphic sequence including a polymorphic site.
  • the polymorphic sequence refers to a sequence including a polymorphic site including SNP in a polynucleotide sequence.
  • the polynucleotide sequence may be DNA or RNA.
  • the SNP marker of the present invention is a marker shown in Tables 1 to 2, and when the polymorphic site of the individual's SNP marker is an allele indicated as a risk allele, the SNP marker may be determined to be a high risk of generating an antibody against a hemophilia therapeutic agent. In the case of an allele labeled as a protective allele, it may be determined that the subject has a low risk of generating an antibody against hemophilia. In the present invention, the "risk allele" can be used interchangeably with the "effect allele.”
  • the SNP marker is an SNP marker for predicting the efficacy of a hemophilia therapeutic agent, and among the polynucleotides derived from the nucleotide sequences of SEQ ID NOs: 1 to 87, in the SNP markers described by SEQ ID NOs: 1 to 84, At least one polynucleotide selected from the group consisting of a base or a polynucleotide consisting of at least 5 consecutive bases comprising the allelic bases of Tables 1 and 2, or a complementary polynucleotide thereof; Can be.
  • SNP may be composed of each of the SNPs, more preferably may be composed of two or more SNPs, most preferably may be composed of all of the SNPs 1 to 87.
  • Efficacy predictive polynucleotide or its complementary polynucleotide of the hemophilia therapeutic agent comprising the SNP according to the present invention is preferably composed of five or more consecutive bases, and more preferably 5 to 50 around the base where the SNP is located Dog, even more preferably 10 to 40, even more preferably 20 to 30 consecutive bases, but is not limited thereto.
  • the present invention provides a composition for predicting efficacy of a hemophilia therapeutic agent, comprising an agent capable of detecting the SNP marker.
  • the detectable agent may be a probe.
  • the probe refers to an oligonucleotide that can be specifically identified through hybridization with a polymorphic site of the gene to predict the efficacy of a hemophilia therapeutic agent.
  • the allele-specific probe of the present invention has a polymorphic site in nucleic acid fragments derived from two individuals of the same species, and hybridizes to a DNA fragment derived from one individual but not to a fragment derived from another individual. In this case, the hybridization conditions should be severe enough to hybridize to only one of the alleles, since the hybridization conditions show a significant difference in hybridization strength between alleles.
  • the central region is preferably aligned with the polymorphic region of the polymorphic sequence. This can lead to good hybridization differences between different allelic forms.
  • the probe of the present invention can be used in diagnostic kits or diagnostic methods such as microarrays for detecting alleles and diagnosing the sensitivity of the hemophilia therapeutic agent. There is no particular limitation on such a gene analysis method, and may be by any gene detection method known in the art.
  • the detectable agent may be a primer.
  • the primer of the present invention refers to a composition capable of predicting the efficacy of a hemophilia therapeutic agent by amplifying a polymorphic site of the gene as described above, and preferably amplifying a polynucleotide of the efficacy predictor marker of the hemophilia therapeutic agent specifically. It means a primer that can be.
  • the primers used to amplify the polymorphic markers can serve as a starting point for template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA polymerase) and appropriate temperatures in appropriate buffers. Refers to a single stranded oligonucleotide.
  • the appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template.
  • the primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.
  • the primer hybridizes to a DNA sequence comprising a polymorphic site to amplify a DNA fragment comprising a polymorphic site.
  • primer is a base sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs, It refers to a short sequence that serves as a starting point for.
  • Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • PCR amplification can be used to predict the production of antibodies to hemophilia by the production of the desired product.
  • sense and antisense primer lengths can be modified based on what is known in the art.
  • Probes or primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, Phosphoramidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
  • the detectable agent may be an aptamer.
  • Aptamers are single-stranded DNA or RNA molecules, and are small single-stranded oligonucleotides capable of specifically recognizing target materials with high affinity. Aptamers have been recognized as alternatives to antibodies because they can be used as a component of biosensors that can recognize molecules in detection and analysis systems. In particular, aptamers can be used as target molecules of various organic and inorganic substances, including toxins, unlike antibodies, and once isolated, the aptamers are reproduced with low cost and consistency by automated oligomer synthesis method. This was possible economically.
  • the present invention provides a kit for predicting efficacy of a hemophilia therapeutic agent comprising the composition for predicting the efficacy of the hemophilia therapeutic agent.
  • the kit may be, but is not limited to, a PCR kit, a DNA analysis kit (eg, a DNA chip) kit, or a fluorescence dye kit.
  • the kit of the present invention can predict the efficacy of the hemophilia treatment agent by confirming the SNP marker, which is an efficacy prediction marker of the hemophilia treatment agent, or by identifying the SNP marker in the amplified cDNA template for mRNA.
  • the kit of the present invention is a genomic DNA derived from a sample to be analyzed to perform PCR, a primer set specific for the marker of the present invention, an appropriate amount of DNA polymerase (eg, Taq polymerase), dNTP It may be a kit containing a mixture, PCR buffer and water.
  • the PCR buffer may contain KCl, Tris-HCl and MgCl 2.
  • the components necessary for performing electrophoresis to confirm amplification of PCR products may be further included in the kit of the present invention.
  • the kit of the present invention may be a kit for predicting the efficacy of a hemophilia therapeutic agent including an element necessary to perform a fluorescent dye kit using a probe type.
  • Analytical methods using anaerobic dyes include TaqMan5 '(nuclease assay), Molecular Beacons, Oligonucleotide Ligase Assay (OLA), Invader Assay (Invasive Cleavage of Oligonucleotide Probes), Pyrosequencing, Single Base Extension, or Fluorescence Polarization. It is not limited to this.
  • the kit of the present invention may be a kit for predicting efficacy of a hemophilia therapeutic agent including an essential element necessary for carrying out a DNA chip.
  • DNA chip kits are those in which a nucleic acid species is attached in a gridded array to a glass surface that is generally no larger than a flat solid support plate, typically a microscope slide.
  • hybridization between the nucleic acid of the phase and the complementary nucleic acid contained in the solution treated on the surface of the chip (hybridization) is a tool that allows massively parallel analysis.
  • the present invention provides an efficacy prediction microarray of a hemophilia therapeutic agent comprising a polynucleotide of the efficacy prediction marker of the hemophilia therapeutic agent.
  • the microarray may include DNA or RNA polynucleotides.
  • the microarray may be formed of a conventional microarray except for including the polynucleotide, polypeptide, cDNA, and the like of the present invention.
  • the efficacy prediction microarray of the hemophilia therapeutic agent is a polynucleotide capable of hybridizing to specifically distinguish bases of polymorphic sites in each polymorphic sequence of SEQ ID NOs: 1 to 87, complementary polynucleotides thereof, or their
  • a probe using a polynucleotide which hybridizes with a polypeptide encoded by it or a cDNA thereof it can be prepared by conventional methods known to those skilled in the art.
  • the polynucleotide is immobilized on a substrate coated with one active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. It is not limited to this.
  • the probe polynucleotide refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid.
  • the probe of the present invention is an allele-specific probe, in which a polymorphic site exists in nucleic acid fragments derived from two members of the same species, hybridizing to a DNA fragment derived from one member, but not to a fragment derived from another member. .
  • hybridization conditions show a significant difference in the hybridization intensity between alleles, and should be strict enough to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms.
  • the probe of the present invention can be used to detect the allele and predict the efficacy of a hemophilia therapeutic agent.
  • the prediction method includes detection methods based on hybridization of nucleic acids such as Southern blot and the like, and may be provided in a form that is pre-bound to the substrate of the DNA chip in a method using a DNA chip.
  • Immobilization of a probe polynucleotide on a substrate associated with predicting the efficacy of a hemophilia therapeutic agent of the invention can also be readily prepared using this prior art.
  • a method of immobilizing the polynucleotide on a substrate it is preferable to use a micropipetting method using a piezoelectric method, a method using a pin type spotter, etc., but not limited thereto. It doesn't work.
  • the detection may, for example, label a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, for example, a substance such as Cy3 and Cy5, and then hybridize on a microarray and from the labeling substance. By detecting the generated signal, the hybridization result can be detected.
  • the present invention provides a method of providing information for predicting the efficacy of a hemophilia therapeutic agent, comprising determining the base of the polymorphic site of the SNP marker.
  • step (a) amplifying a polymorphic site of the SNP marker from the DNA of the separated sample or hybridize with the probe; And (b) determining a base of the amplified or hybridized polymorphic site of step (a), and may provide a method for providing information for predicting the efficacy of the hemophilia therapeutic agent.
  • DNA of the isolated sample can be obtained from a sample separated from the individual.
  • DNA may be obtained from a sample such as hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva, but is not limited thereto.
  • Amplifying the polymorphic site of the SNP marker from the DNA of step (a) or hybridizing with the probe may be any method known to those skilled in the art.
  • the target nucleic acid can be amplified by PCR and purified.
  • Other ligase chain reactions (LCRs), transcription amplifications and self-sustained sequence replications and nucleic acid based sequence amplifications (NASBAs) can be used.
  • Determining the base of the polymorphic site of step (b) of the method is sequence analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization technique , DASH), PCR prolongation analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (eg Sequenom's MassARRAY system), mini-sequencing method, Bio- Plex Systems (BioRad), CEQ and SNPstream Systems (Beckman), Molecular Inversion Probe Array Technologies (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium Assays) .
  • One or more alleles in the polymorphic markers can be identified by the above methods or other methods available to those skilled in the art. Determining the base of such a polymorphic site can be preferably performed through the SNP chip.
  • SNP chip refers to one DNA microarray capable of identifying each base of several hundred thousand SNPs at once.
  • TaqMan methods include the steps of: (1) designing and constructing primers and TaqMan probes to amplify desired DNA fragments; (2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems); (3) using the DNA as a template and performing PCR using the primers and probes described above; (4) after the PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotides of step (1) from the analysis result.
  • allele-specific PCR refers to a PCR method of amplifying a DNA fragment in which the SNP is located with a primer set including a primer designed with the base where the SNP is located as the 3 'end.
  • PCR extension assays first inactivate DNA fragments containing bases on which a single nucleotide polymorphism is located by amplification with primer pairs and then dephosphorylation of all nucleotides added to the reaction, followed by SNP specific extension primers, dNTP mixtures.
  • SNP specific extension primers dNTP mixtures.
  • the extension primer is a 3 'end of the base immediately adjacent to the 5' direction of the base where the SNP is located
  • the dNTP mixture excludes a nucleic acid having the same base as the didioxynucleotide, and the didioxynucleotide represents the SNP.
  • the SNP when fluorescently labeling an extension primer or didioxynucleotide, the SNP can be detected by detecting fluorescence using a gene analyzer (for example, Model 3700, manufactured by ABI, etc.) used for determining a general sequence.
  • a gene analyzer for example, Model 3700, manufactured by ABI, etc.
  • the SNP can be detected by measuring molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique.
  • MALDI-TOF matrix assisted laser desorption ionization-time of flight
  • rs2486316 as set forth in SEQ ID NO: 1, wherein the 101st base is T;
  • the rs476488 set forth in SEQ ID NO: 2, wherein the 101 st base is C;
  • the rs55700376 set forth in SEQ ID NO: 3, wherein the 101 rd base is A;
  • the rs1131568 set forth in SEQ ID NO: 4, wherein the 101 st base is T;
  • the rs3815496 set forth in SEQ ID NO: 5, wherein the 101 st base is C;
  • the rs62334652 set forth in SEQ ID NO: 6, wherein the 101 st base is C;
  • the rs45468202 set forth in SEQ ID NO: 7, wherein the 101 st base is C;
  • the rs3805220 set forth in SEQ ID NO: 8, wherein the 101 st base is A;
  • the rs13306436 set forth in SEQ ID NO: 9, wherein the
  • the polymorphic site may be determined as a risk allele, and the higher the frequency of the allele, the higher the risk of generating a hemophilia therapeutic antibody. More preferably, when two or more polymorphic sites of SEQ ID NOs: 1 to 26, 58 to 71, and 85 to 87 are risk alleles, most preferably, all 43 are alleles that are at higher risk for hemophilia therapeutic antibody production. Can be. When the 101 st base is not the base of the risk allele of Table 1, it can be determined that the risk of generating hemophilia therapeutic antibody.
  • SNPs in Table 1 (OR> 1) increase the risk of antibody production against hemophilia therapeutics.
  • rs366316 described in SEQ ID NO: 27 among the base sequences determined in step (b), when the 101 st base is G;
  • the rs3213479 set forth in SEQ ID NO: 29, wherein the 101 st base is A;
  • the rs17141154 set forth in SEQ ID NO: 30, wherein the 101 st base is A;
  • the rs2269443 set forth in SEQ ID NO: 31 wherein the 101 st base is A;
  • the rs2459467 set forth in SEQ ID NO: 32, wherein the 101 st base is T;
  • the rs2230525 set forth in SEQ ID NO: 33, wherein the 101 st base is C;
  • the rs6876997 set forth in SEQ ID NO: 34, wherein the 101 st base is G;
  • the rs149956505 set forth
  • the polymorphic site is determined as a protective allele, and the higher the frequency of the allele, the lower the risk of antibody production of the hemophilia treatment agent. More preferably, when two or more polymorphic sites of SEQ ID NOs: 27 to 57 and 72 to 84 are protective alleles, most preferably, all 44 protective alleles have a lower risk of generating hemophilia A therapeutic antibody. When the 101 st base is not the base of the protective allele of Table 2, it can be determined that the risk of generating hemophilia therapeutic antibody.
  • SNPs in Table 2 reduce the risk of antibody production against hemophilia therapeutics.
  • the present invention is to provide a method for selecting the SNP predictive efficacy of a hemophilia therapeutic agent.
  • the sequencing depth value of 10 or more and the genotype quality value of 20 or more in the whole length exome sequencing of the subject to which hemophilia treatment is administered Screening SNPs and removing intergenic variants that do not have residual regions around them; (b) performing an associative analysis on the SNPs selected in step (a) to select the SNPs associated with the hemophilia therapeutic antibody antibody phenotype; And (c) selecting the SNPs of the gene regions related to the immune system with respect to the SNPs selected in step (b).
  • the term “Sequencing Depth” refers to the number of bases read during the sequencing process.
  • the sequencing depth (range) indicates how many times the total number read is greater than the length of the sequence being analyzed, and the depth is the average number of reads representing a particular base in the reconstructed sequence.
  • the term Genotype Quality (GQ) is also a Phred-scaled value indicating the confidence that the genotype is a true genotype. This higher value reflects a more accurate genotype.
  • Whole exome sequencing in step (a) can be made on a DNA sample of a subject undergoing hemophilia drug treatment using a SureSelect XT v5 Kit and performed on a NextSeq 500 instrument.
  • Intergene modification means an SNP in which a gene region does not exist around the SNPs selected by performing the full-length exome sequencing.
  • the correlation analysis was performed by Fisher's Exact Test, which is a method of testing the frequency data constituting the partition table by using the initial load probability distribution.
  • the correlation analysis is performed for the entire population, the F8 inversion detection group, respectively, and the comparison of the groups for the antibody production response is> 0.60 BU group vs. ⁇ 0.60 BU group and> 5.00 BU group vs. ⁇ It was divided into 5.00 BU groups.
  • OR value used in the present invention is an odds ratio, which represents a ratio calculated as an estimate of relative risk in a case-control study, and an example of a probability of having a corresponding SNP in a control group. It shows the ratio of the probability of having the corresponding SNP in the group. For example, if a group of subjects exposed to risk factors, b unexposed members, c exposed controls, and non-exposed members, the odds ratio would be ad / bc.
  • the odds ratio is greater than 1, the corresponding multi-SNP is associated with the production of antibodies to hemophilia treatment agents, and when the odds ratio is less than 1, it means that the multi-SNPs are not associated with the production of antibodies to hemophilia A therapeutic agents.
  • the larger the odds ratio the higher the association.
  • the smaller the odds ratio the smaller the association.
  • results of the SNP polymorphism can be statistically processed using a statistical analysis method commonly used in the art, for example, Student's t-test, Chi-square test (Chi- Continuous variables, categorical variables, and 95% confidence intervals obtained through square test, linear regression line analysis, multiple logistic regression analysis, etc. Can be analyzed using variables such as
  • step (c) the SNPs of the gene region related to the immune system are selected with respect to the SNPs selected in step (b), and preferably, HGVS (Human Genome Variation Society) information of the SNPs is checked.
  • the steps of (a) to (c) were performed to select 87 SNP marker sets (four duplicate SNPs) for predicting the efficacy of the hemophilia therapeutic agent.
  • Example 1 Test of Antibody Production Response of Test Specimen and Hemophilia Therapeutics
  • DNA samples from 250 hemophilia patients were prepared using the SureSelect XT v5 Kit and a whole exome sequencing was performed on the NextSeq 500. Was performed.
  • SNPs with repeated statistical significance were selected.
  • SNPs with an OR value of> 1 have a high risk of generating antibodies against hemophilia A treatment, whereas SNPs with an OR value of ⁇ 1 have a low risk of producing antibodies against hemophilia A treatment.
  • SNPs having an OR value of> 5 or ⁇ 0.2 may be considered to have a statistically strong association with the antibody producing response.
  • the OR values for the antibody-producing groups were> 5 or ⁇ 0.2 and P ⁇ 0.05.
  • the predictive efficacy of the hemophilia treatment agent may be used to predict an objective risk of generating antibodies against the hemophilia treatment agent.

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Abstract

La présente invention concerne un marqueur permettant de prédire l'efficacité d'agents thérapeutiques contre l'hémophilie et, plus particulièrement, un marqueur de polymorphisme mononucléotidique (SNP) permettant de prédire l'efficacité d'agents thérapeutiques contre l'hémophilie, une composition comprenant un agent capable de détecter le marqueur de SNP, un kit ou une puce à ADN comprenant la composition, un procédé permettant de fournir des informations sur la prédiction de l'efficacité des agents thérapeutiques contre l'hémophilie à l'aide du marqueur de SNP, et un procédé de criblage de SNP permettant de prédire l'efficacité d'agents thérapeutiques contre l'hémophilie.
PCT/KR2017/006344 2016-06-23 2017-06-16 Marqueur de prédiction de l'efficacité d'un agent thérapeutique contre l'hémophilie et son utilisation Ceased WO2017222247A2 (fr)

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KR1020160078447A KR101774996B1 (ko) 2016-06-23 2016-06-23 혈우병 치료제의 효능 예측 마커 및 이의 용도
KR10-2016-0078447 2016-06-23

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