WO2023248997A1 - Single nucleotide polymorphism haplotype analysis method in human sting1 gene - Google Patents

Single nucleotide polymorphism haplotype analysis method in human sting1 gene Download PDF

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WO2023248997A1
WO2023248997A1 PCT/JP2023/022685 JP2023022685W WO2023248997A1 WO 2023248997 A1 WO2023248997 A1 WO 2023248997A1 JP 2023022685 W JP2023022685 W JP 2023022685W WO 2023248997 A1 WO2023248997 A1 WO 2023248997A1
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seq
snps
sequencing
combination
primer pair
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淳 村井
武史 大坪
史晴 横山
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小野薬品工業株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • 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
    • C12Q1/6869—Methods for sequencing
    • 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

Definitions

  • the present invention relates to a haplotype analysis method for single nucleotide polymorphisms (hereinafter sometimes abbreviated as "SNP”) in the human STING1 gene.
  • SNP single nucleotide polymorphisms
  • STING Stimulation of Interferon Genes
  • cGAS cyclic GMP-AMP synthase
  • cGAMP cyclic GMP-AMP
  • cyclic dinucleotides such as cyclic Di-GMP, which was first identified as a second messenger in bacteria and later confirmed to exist in mammals, are also known to directly bind to and activate STING (Non-patent literature 1).
  • STING is also known to be involved in autoimmune diseases and tumor immunity.
  • abnormal host DNA has been shown to leak from the nucleus and activate STING, inducing a pro-inflammatory response, which has been implicated in autoimmune diseases.
  • the STING pathway also detects tumor-derived DNA and promotes T cell responses against tumors.
  • STING agonist compounds administered to mouse tumors induce adaptive immune responses leading to tumor regression (Non-Patent Document 2), and molecules activating the STING pathway enhance IFN production and exhibit antiviral effects.
  • Non-Patent Document 3 is known, and various compounds that actuate STING have already been reported (Non-Patent Document 4).
  • An object of the present invention is to provide a method for determining haplotypes based on a combination of multiple SNPs in the human STING1 gene.
  • the present invention provides a method for determining a haplotype based on a combination of four SNPs in the human STING1 gene.
  • the present invention makes it possible to determine a haplotype based on a combination of four SNPs in the human STING1 gene.
  • the present invention mainly relates to an analysis method for determining a haplotype based on a combination of SNPs each specified by rs7380824, rs1131769, rs78233829, and rs11554776 in the human STING1 gene, and includes the following configuration. That is, (a) Extracting each of genomic DNA and RNA from a human biological sample; (b) From the RNA extracted in the step described in (a) above, complementary DNA (hereinafter referred to as "cDNA”) is generated by a reverse transcription reaction (hereinafter sometimes abbreviated as "RT reaction”). (sometimes abbreviated as complementary DNA).
  • a step of performing PCR (Polymerase Chain Reaction) separately under predetermined reaction conditions for each primer pair using one or more primer pairs designed to sandwich the (d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof; (e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, A step of separately performing PCR under predetermined reaction conditions; (f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above.
  • PCR Polymerase Chain Reaction
  • step (f) From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. Complementing the information includes determining the haplotypes of both alleles of the human STING1 gene. An overview of the analysis method is shown in Figure 1.
  • the human STING1 gene according to the present invention is a gene encoding human STING or its isoform specified by National Center for Biotechnology Information (NCBI) Reference Sequence: NP_938023.1 (SEQ ID NO: 37), and NCBI Identified by Entrez Gene: 340061.
  • NCBI National Center for Biotechnology Information
  • NP_938023.1 SEQ ID NO: 37
  • NCBI NCBI Identified by Entrez Gene: 340061.
  • Single nucleotide polymorphism also called “SNP” ( Single Nucleotide Polymorphism ), refers to a polymorphism caused by a single nucleotide substitution in the base sequence of genomic DNA.
  • rs1131769 represents a cytosine and thymine polymorphism (corresponding to guanine and adenine polymorphisms, respectively, on complementary strand genomic DNA) at the 139478334th base on human chromosome 5, and the base is
  • the base is cytosine
  • the 232nd amino acid of the human STING is wild-type arginine
  • the amino acid is histidine
  • the polymorphism of this amino acid is expressed as "R232H”.
  • rs78233829 represents a cytosine and guanine polymorphism (corresponding to guanine and cytosine polymorphisms, respectively, on complementary strand genomic DNA) at the 139478340th base of human chromosome 5, and the base is
  • the 230th amino acid of the human STING is the wild type glycine
  • the amino acid is alanine
  • the polymorphism of this amino acid is designated as "G230A”. Note that this SNP is adjacent to the SNP of rs1131769 described above.
  • rs11554776 represents a cytosine and thymine polymorphism (corresponding to guanine and adenine polymorphisms, respectively, on complementary strand genomic DNA) at the 139481493rd base on human chromosome 5, and the base is
  • the base is cytosine, it is wild type, and the 71st amino acid of the human STING is arginine; when the base is thymine, the amino acid is histidine, and the polymorphism of this amino acid is expressed as "R71H".
  • haplotype Name the entries in the vertical column of the column labeled "Haplotype Name" represent the abbreviation of each haplotype.
  • Each letter of the alphabet written horizontally in these columns represents the abbreviation of the amino acid corresponding to each codon having the four SNPs. Note that determining such haplotypes is called haplotyping.
  • Chromosomes in principle, have homologous genes consisting of a combination of two alleles, so the haplotype that an individual has consists of one set derived from each allele. Based on the haplotypes, there are, for example, the combinations shown in Table 2.
  • RAHR at the bottom of the right column in Table 2 represents a haplotype in which the 71st position of human STING is arginine, the 230th position is alanine, the 232nd position is histidine, and the 293rd position is arginine.
  • the "biological sample” in the present invention may be a part of any tissue collected from a subject, but blood, in particular nucleated cells, from which collection and extraction of DNA and RNA are relatively easy. Whole blood containing blood is preferred.
  • the blood sample may be added with an anticoagulant (eg, sodium citrate, etc.) used during blood collection, or may be blood sampled using an EDTA blood collection tube. Further, the blood sample may be one that has been stored at low temperature or frozen.
  • an anticoagulant eg, sodium citrate, etc.
  • Extraction of genomic DNA from biological samples can be carried out by methods known to those skilled in the art, such as methods using thermal extraction, alkaline thermal extraction, phenol/chloroform extraction, spin column methods, magnetic bead methods, etc.
  • methods using thermal extraction for example, NucleoSpin (registered trademark) RNA Blood and NucleoSpin (registered trademark) RNA/DNA Buffer Set (MACHEREY-NAGEL), etc.
  • RNA extraction kit for example, NucleoSpin (registered trademark) RNA Blood and NucleoSpin (registered trademark) RNA/DNA Buffer Set (MACHEREY-NAGEL), etc.
  • the RT reaction refers to a reaction in which cDNA is synthesized from mRNA (messenger RNA) using a reverse transcriptase (eg, PrimeScript (registered trademark) RTase).
  • cDNA is DNA that is produced by reverse transcriptase using mRNA as a template and has a base sequence complementary to mRNA.
  • the RT reaction consists of a reaction in which an oligo dT primer is annealed to mRNA, a reverse transcription reaction in which the oligo dT primer is extended using mRNA as a template, and a reaction in which reverse transcriptase is inactivated by high temperature, and can be carried out by methods known to those skilled in the art. For example, there is a method using a commercially available kit (eg, PrimeScript RT-PCR kit (Takara Bio), etc.).
  • PCR Polymerase Chain Reaction
  • the reaction (i) denatures the targeted DNA, (ii) anneals the primer to its primer binding site, and (iii) injects a thermostable DNA polymerase (e.g. , Taq DNA polymerase, etc.) can be repeated multiple times.
  • a thermostable DNA polymerase e.g. , Taq DNA polymerase, etc.
  • the reaction is performed using a thermal cycler at an optimized temperature, duration, and number of cycles; the conditions are, for example, as described by McPherson et al, PCR 2: A Practical Approach (July 27, 1995 by Oxford). It can be set in advance by referring to the University.
  • the information can be used with existing primer design software, such as OLIGO® Primer Analysis Software (Molecular Biology Insights), Primer3 (https://primer3.ut.ee/) or Primer-BLAST (https:// It can be designed using tools such as www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi).
  • primer design software such as OLIGO® Primer Analysis Software (Molecular Biology Insights), Primer3 (https://primer3.ut.ee/) or Primer-BLAST (https:// It can be designed using tools such as www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi).
  • Sequencing or sequencing in the present invention refers to a method for determining the base sequence of DNA, and can be carried out by methods known to those skilled in the art, such as Sanger sequencing (dideoxy sequencing) (Proc Natl. Acad Sci U S A. 1977 Dec; 74(12): 5463-5467) and Maxam-Gilbert sequencing (Proc Natl Acad Sci U S A. 1977 February; 74(2): 560-564). Furthermore, next generation sequencing ( NGS ), e.g.
  • the SNP genotype according to the present invention refers to the combination of bases in a specific SNP between two alleles.
  • the genotype of the SNP specified in rs7380824 is expressed as “CC” if the bases at the same locus on two alleles are both cytosine, and “CT” if they are cytosine and thymine. and both are thymine, it is expressed as “TT”.
  • genotypes of the SNP identified by rs1131769 include “CC”, “CT”, and “TT”
  • genotypes of the SNP identified by rs78233829 include “CC”, “CG”, and There is “GG”
  • SNP genotypes specified at rs11554776 include “CC”, “CT”, and “TT”. Note that determining such a genotype is called genotyping.
  • the RT reaction in the step (b) above is performed, for example, after an annealing reaction (RNA extract is held at 65°C for 5 minutes and then held at a low temperature (e.g., 4°C)). , for 30 minutes at 42°C, followed by 15 minutes at 70°C, and then held at 4°C.
  • a low temperature e.g. 4°C
  • each of the primer pairs (c1) to (c3) is preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NO: 1 and 2, and a pair of base sequences of SEQ ID NO: 7 and 8, respectively. and a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 13 and 14.
  • PCR in the step described in (c) above can be carried out, for example, under the conditions shown in Table 3, but the annealing step with the primers is performed at about 55 to about 65°C. It can also be carried out under conditions of holding for about 15 to 60 seconds.
  • the sequencing in the step (d) above can be performed, for example, by next-generation sequencing, and is preferably performed by synthetic sequencing (for example, MiSeq (registered trademark) (Illumina)). It can be carried out using the method used in this study.
  • synthetic sequencing for example, MiSeq (registered trademark) (Illumina)
  • the primer pair sandwiching one or more SNPs among the four SNPs in the steps (e) and (ii) above may be, for example, (e1) A primer pair sandwiching the SNP of rs7380824 (for example, from a set of base sequences selected from (i) SEQ ID NOs: 19 and 20, (ii) SEQ ID NOs: 21 and 22, and (iii) SEQ ID NOs: 23 and 24) a combination of single-stranded DNA), (e2) A primer pair sandwiching the two SNPs rs1131769 and rs78233829 (for example, a pair selected from (i) SEQ ID NOs: 25 and 26, (ii) SEQ ID NOs: 27 and 28, and (iii) SEQ ID NOs: 29 and 30) A combination of single-stranded DNA consisting of the nucleotide sequence of A combination of single-stranded DNA consisting of a set of base sequences selected from numbers 35 and 36)
  • Each of the primer pairs (e1) to (e3) is preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 21 and 22, and a pair of base sequences of SEQ ID NOs: 27 and 28, respectively. and a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 33 and 34.
  • PCR in the step described in (e) above can be carried out under the same conditions as shown in Table 3, but the annealing step with the primers can be carried out at 55 to 65°C. It can also be carried out under conditions of holding for about 15 to 60 seconds.
  • the sequencing in the step (f) above can be performed, for example, by Sanger sequencing.
  • the haplotype analysis method of the present invention from the DNA sequence information obtained in the step described in (d) above in the step described in (g) (i) above, the four SNPs are determined for both alleles of the human STING1 gene.
  • methods for extracting haplotypes based on combinations include the following methods.
  • Vcflib version 1.0.2, GitHub, https://github.com/vcflib/vcflib
  • VCFtools version 0.1.16, GitHub, https://github.com/vcftools/vcftools
  • bcftools version 1.12, GitHub, https:// github.com/samtools/bcftools
  • haplotype phasing of the detected measured SNPs using WhatsHap (version 1.0, GitHub, https://github.com/whatshap/whatshap/) and merged the separate vcf files using bcftools. to obtain haplotype results.
  • a method for extracting the genotype at each of the four SNPs from the DNA sequence information obtained in the step (f) above in the step (g) (ii) above For example, the following method may be mentioned.
  • the haplotype of the SNP in one allele can be derived by adding separately acquired information regarding the genotype for each SNP to the information regarding the haplotype.
  • the information regarding the haplotype extracted in the step (i) above is converted to the genotype extracted in the step (ii) above.
  • methods for determining the haplotypes of both alleles of the human STING1 gene by supplementing with information on the STING1 gene include the following method. ( ⁇ ) If two haplotypes are confirmed and match the genotype results, the obtained haplotype is determined as the final result. ( ⁇ ) If one haplotype is confirmed and the genotype is heterozygous, one haplotype can be created by subtracting one allele obtained by haplotyping from the two alleles obtained by genotyping. Confirm.
  • genotype If the genotype is homozygous in the entire region and the same haplotype is obtained, it is determined to be homozygous for the obtained type. ( ⁇ ) If there is a discrepancy between the genotyping results and haplotyping results, the genotype is undetermined.
  • step (f) From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above.
  • the method comprises determining the haplotypes of both alleles of the human STING1 gene by supplementing the information; [2] The method according to [1] above, wherein the human biological sample is whole blood; [3] The one or more primer pairs described in the preceding paragraph [1] (c) (ii) are (a) primer pairs that sandwich all of the four SNPs, (b) primers that sandwich the SNPs of rs7380824, rs1131769, and rs78233829.
  • the primer pair sandwiching the SNP of rs7380824 described in [12] (a) above is selected from (i) SEQ ID NO: 19 and 20, (ii) SEQ ID NO: 21 and 22, and (iii) SEQ ID NO: 23 and 24.
  • the method according to [12] above which is a combination of single-stranded DNA consisting of a set of base sequences; [14]
  • the primer pair sandwiching the two SNPs rs1131769 and rs78233829 described in the previous section [12] (b) is (i) SEQ ID NO: 25 and 26, (ii) SEQ ID NO: 27 and 28, and (iii) SEQ ID NO: 29.
  • step (f) From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above.
  • the process consists of determining the haplotypes of both alleles of the human STING1 gene by supplementing with information,
  • the RT reaction described in (b) above is carried out under the conditions shown above (extracted RNA is kept at about 65°C for about 5 minutes, then held at about 4°C, then at about 42°C for about 30 minutes, followed by a temperature of about 70°C for about 15 minutes, followed by a hold at about 4°C)
  • the one or more primer pairs described in (c)(ii) above are (a) a primer pair sandwiching all four SNPs (preferably a single strand consisting of a pair of nucleotide sequences SEQ ID NOs: 1 and 2); (a combination of DNA), (b) a primer pair sandwiching SNPs of rs7380824, rs1131769 and rs78233829 (preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 7 and 8), and (c)
  • a primer pair sandwiching two SNPs, rs1131769 and rs78233829 (preferably, a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 27 and 28)
  • a primer pair (preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 33 and 34) sandwiching the SNP of rs11554776,
  • the PCR described in (e) above is carried out under the conditions shown in Table 3 above, The method, wherein the sequencing described in (f) above is Sanger sequencing; [1-1] Single-stranded DNA consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 18; and [1-2] Single strand DNA consisting of a base sequence selected from the group consisting of SEQ ID NOS: 19 to 36 Strand DNA.
  • Example 1 As a pretreatment for genomic DNA extraction from blood samples , an equal amount of Lysis Buffer DL (a reagent included in the NucleoSpin RNA/DNA Buffer Set (Macherey-Nagel)) was added to frozen blood, Melted and dissolved. 800 ⁇ L of the equal volume of the mixed solution was dispensed into a 2.0 mL tube. 10 ⁇ L of Proteinase K solution (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was added, and the mixture was shaken vigorously for 15 minutes at room temperature (18-25°C). 400 ⁇ L of 70% ethanol was added to the same reaction solution and mixed.
  • Lysis Buffer DL a reagent included in the NucleoSpin RNA/DNA Buffer Set (Macherey-Nagel)
  • 800 ⁇ L of the equal volume of the mixed solution was dispensed into a 2.0 mL tube. 10 ⁇ L of Proteinase K solution (a reagent included in the
  • the yield and quality of the extracted DNA was confirmed. Fluorescence quantification was performed using a Qubit Fluorometer (Thermo Fisher Scientific), and the Qubit concentration was confirmed to be 600 ng or more (concentration 10 ng/ ⁇ L or more).
  • Example 2 Extraction of Total RNA from Blood Samples 800 ⁇ L of an equal volume mixture of pretreated blood/Lysis Buffer DL in the same manner as in Example 1 was dispensed into a 2.0 mL tube. 10 ⁇ L of Proteinase K solution was added, and the mixture was shaken vigorously for 15 minutes at room temperature (18-25°C). 400 ⁇ L of 70% ethanol was added to the same reaction solution and mixed. The same mixture was added to a NucleoSpin RNA Blood Column and centrifuged to adsorb the nucleic acid onto the silica membrane. 350 ⁇ L of Membrane Desalting Buffer (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was added to the column to desalt the membrane.
  • Membrane Desalting Buffer a reagent included in the NucleoSpin RNA/DNA Buffer Set
  • the primer pair that sandwiched the SNP of rs7380824 consisted of SEQ ID NOs: 21 and 22, and the primer pair that sandwiched the two SNPs of rs1131769 and rs78233829 consisted of SEQ ID NOs: 27 and 28, respectively.
  • Primers consisting of SEQ ID NOs: 33 and 34 were used as primer pairs sandwiching the SNP of rs11554776 (FIG. 3).
  • Example 2 To the DNA solution prepared in Example 1 (containing 100 ng of DNA), 25 ⁇ L of 2 ⁇ Gflex PCR Buffer (Mg 2+ , dNTP plus) (Takara Bio Inc.) and 10 pmol each of the above primer pair solutions (forward primer and reverse primer) were added. The corresponding amount and 1 ⁇ L of Tks Gflex DNA Polymerase (1.25 units/ ⁇ L) (Takara Bio Inc.) were mixed, Nuclease Free water was added to make the total volume 50 ⁇ L, and the mixture was mixed to prepare each PCR reaction solution.
  • 2 ⁇ Gflex PCR Buffer Mg 2+ , dNTP plus
  • 10 pmol each of the above primer pair solutions forward primer and reverse primer
  • PCR reaction solutions were set in a Veriti 200 thermal cycler (Thermo Fisher Scientific), and PCR was performed under the PCR conditions shown in Table 4.
  • PCR products were purified using AMPure XP (Beckman Coulter), and the concentration and peak size were confirmed using Agilent 2200/4200 TapeStation. It was confirmed that there was no deviation of % or more, that there was a single peak, and (ii) that the TapeStation concentration was 20 ng or more and the concentration was 1 ng/ ⁇ L or more.
  • the primer pairs sandwiching the four SNPs were the primers consisting of SEQ ID NOs: 1 and 2, and the primer pairs sandwiching the SNPs of rs7380824, rs1131769, and rs78233829 were SEQ ID NOs: 7 and 2, respectively.
  • Primers consisting of SEQ ID NOs: 13 and 14 were used as primer pairs sandwiching the SNPs of rs1131769, rs78233829, and rs11554776, respectively (FIG. 2).
  • the PCR reaction solution was set in a Veriti 200 thermal cycler, and PCR was performed under the PCR conditions shown in Table 4 above.
  • Each of the PCR products was purified using AMPure XP (Beckman Coulter), and concentration and peak size were confirmed using Agilent 2200/4200 TapeStation to confirm yield and quality. As a result, it was confirmed that (i) a peak of the target PCR amplification product excluding Primer Dimer could be confirmed, and (ii) the TapeStation concentration was 40 ng or more and the concentration was 1 ng/ ⁇ L or more.
  • each PCR product (each PCR product of region 5, region 6, and region 7) in Elution Buffer (Buffer EB, QIAGEN) so that the concentration is the same, and add each PCR product of region 5, region 6, and region 7. were mixed in a ratio of 2:1:1.
  • Elution Buffer Buffer EB, QIAGEN
  • the mixture was placed in a Veriti 200 thermal cycler and the reaction was started (25 minutes at 22°C, then 20 minutes at 55°C, then held at 4°C).
  • the reaction product was purified using AMPure XP, and the amplified peak size and concentration were measured using D1000 Screen Tape, D1000 Reagent Kit, and Agilent 2200/4200 TapeStation.
  • the main peak of the library was confirmed to have the expected PCR amplification length, and the library concentration was confirmed to be 2 nM or more.
  • Example 8 High-speed sequencing using Miseq System Sequence analysis of the library prepared in Example 7 was performed using Miseq System (Illumina).
  • the prepared library and PhiX Control v3 were mixed at a ratio of 3:2, and 300 base both-end sequence analysis was performed using Miseq System (Illumina).
  • the nucleotide sequence (read sequence) was obtained using the software included with the sequencer (MiSeq Control Software v4.0.0.1769, Real Time Analysis v1.18.54.4, bcl2fastq2v2.17).
  • Example 9 Detection of haplotypes of SNPs in the STING1 gene
  • the nucleotide sequence data obtained by the Miseq System was mapped to the human genome reference sequence GRCh38 (GENCODE) using STAR (version 2.7.9a), and was mapped to the human genome reference sequence GRCh38 (GENCODE) using FreeBayes (version 1.3.5).
  • Haplotype mutation detection analysis was performed using . Bases with measured SNPs of QV40 or higher were used for analysis, and the heterocall threshold was set to Allele frequency > 0.1.
  • Vcflib version 1.0.2
  • VCFtools version 0.1.16
  • bcftools version 1.12
  • Haplotype phasing of the detected measured SNPs was performed using WhatsHap (version 1.0), and separate vcf files were merged using bcftools (version 1.12) to obtain haplotype results.
  • the haplotype analysis method for the human STING1 gene of the present invention is useful for selecting patients who require treatment with STING agonists.

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Abstract

The problem addressed by the present invention is to provide a method for determining haplotype based on a combination of multiple single nucleotide polymorphisms (SNP) in the human STING1 gene.　As a result of in-depth studies by the present inventors, such a method was established, and the present invention was completed. Specifically, the present invention provides a method for determining haplotype based on the combination of four SNP each identified by rs7380824, rs1131769, rs78233829, and rs11554776 in the human STING1 gene.

Description

ヒトＳＴＩＮＧ１遺伝子における一塩基多型のハプロタイプ解析法Haplotype analysis method for single nucleotide polymorphism in human STING1 gene
　本発明は、ヒトSTING1遺伝子における一塩基多型（以下、「SNP」と略記することがある。）のハプロタイプ解析法に関する。 The present invention relates to a haplotype analysis method for single nucleotide polymorphisms (hereinafter sometimes abbreviated as "SNP") in the human STING1 gene.
　STING（Stimulation of Interferon Genes）は小胞体局在型の４回膜貫通型タンパク質であり、自然免疫に関与していることが知られている。感染などにより細胞質に異質の二本鎖DNAが出現すると、環状GMP-AMP合成酵素（cGAS）が活性化され、環状GMP-AMP（cGAMP）が合成される。このcGAMPが小胞体のSTINGと結合し、Ｉ型インターフェロン（IFN）の産生を誘導する。一方、細菌のセカンドメッセンジャーとして最初に同定され、後に哺乳動物でも存在が確認された環状Di-GMPなどの環状ジヌクレオチドも直接STINGに結合し、活性化させることが知られている（非特許文献１）。 STING (Stimulation of Interferon Genes) is a four-transmembrane protein localized in the endoplasmic reticulum and is known to be involved in innate immunity. When foreign double-stranded DNA appears in the cytoplasm due to infection, etc., cyclic GMP-AMP synthase (cGAS) is activated and cyclic GMP-AMP (cGAMP) is synthesized. This cGAMP binds to STING in the endoplasmic reticulum and induces the production of type I interferon (IFN). On the other hand, cyclic dinucleotides such as cyclic Di-GMP, which was first identified as a second messenger in bacteria and later confirmed to exist in mammals, are also known to directly bind to and activate STING (Non-patent literature 1).
　さらにSTINGは、自己免疫疾患や腫瘍免疫にも関与することも知られている。例えば、異常な宿主DNAが核から漏出してSTINGが活性化され、炎症促進性応答を誘導することが示されており、自己免疫疾患への関与が示されている。また、STING経路は、腫瘍由来DNAを検出し、腫瘍に対するＴ細胞応答を促進する。マウスの腫瘍に投与されたSTING作動化合物は、獲得免疫応答を誘導して腫瘍退縮をもたらすこと（非特許文献２）、STING経路の活性化分子がIFN産生を増強し、抗ウイルス作用を示すこと（非特許文献３）が知られており、既に、STINGを作動させる化合物が種々報告されている（非特許文献４）。 Furthermore, STING is also known to be involved in autoimmune diseases and tumor immunity. For example, abnormal host DNA has been shown to leak from the nucleus and activate STING, inducing a pro-inflammatory response, which has been implicated in autoimmune diseases. The STING pathway also detects tumor-derived DNA and promotes T cell responses against tumors. STING agonist compounds administered to mouse tumors induce adaptive immune responses leading to tumor regression (Non-Patent Document 2), and molecules activating the STING pathway enhance IFN production and exhibit antiviral effects. (Non-Patent Document 3) is known, and various compounds that actuate STING have already been reported (Non-Patent Document 4).
　ところで、ヒトSTING1遺伝子には、STINGの応答に影響を及ぼす可能性のある少なくとも４種のSNPが存在することが報告されており、それらSNPの解析法が報告されている（特許文献１）。 Incidentally, it has been reported that the human STING1 gene has at least four types of SNPs that may affect the STING response, and a method for analyzing these SNPs has been reported (Patent Document 1).
国際公開第２０２１／２５０５３０号パンフレットInternational Publication No. 2021/250530 pamphlet
　本発明の課題は、ヒトSTING1遺伝子における複数のSNPの組み合わせに基づくハプロタイプを決定するための方法を提供することにある。 An object of the present invention is to provide a method for determining haplotypes based on a combination of multiple SNPs in the human STING1 gene.
　本発明の発明者らは鋭意検討した結果、同法を確立して、本発明を完成した。すなわち、本発明は、ヒトSTING1遺伝子における４個のSNPの組み合わせに基づくハプロタイプを決定するための方法を提供する。 As a result of intensive study, the inventors of the present invention established the method and completed the present invention. That is, the present invention provides a method for determining a haplotype based on a combination of four SNPs in the human STING1 gene.
　本発明により、ヒトSTING1遺伝子における４個のSNPの組み合わせに基づくハプロタイプを決定することが可能となる。 The present invention makes it possible to determine a haplotype based on a combination of four SNPs in the human STING1 gene.
本発明のハプロタイプの解析法の概要を表す。1 shows an overview of the haplotype analysis method of the present invention. 下記（ｃ）（ｉｉ）記載の工程における、４個のSNP（rs7380824、rs1131769、rs78233829およびrs11554776）のうち、隣接して連続する少なくとも３個のSNPを挟むように設計されたプライマーの組合せ（以下、「プライマーの組合せ」を「プライマーペア」と記載することがある。）を表す。表中、「方向」は、プライマーの方向を表し、「Ｆ」はForward方向を、「Ｒ」はReverse方向を各々表し、「SNP」はプライマーペアによって挟まれるSNPを表す。In the step described in (c) (ii) below, a combination of primers (the following , a "combination of primers" is sometimes referred to as a "primer pair"). In the table, "Direction" represents the direction of the primer, "F" represents the forward direction, "R" represents the reverse direction, and "SNP" represents the SNP sandwiched between the primer pairs. 下記（ｅ）（ｉｉ）記載の工程における、当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを表す。表中の「Ｆ」、「Ｒ」および「SNP」は各々図２と同じ意味を表す。It represents a pair of primers each sandwiching one or more SNPs among the four SNPs in the steps described in (e) and (ii) below. "F", "R" and "SNP" in the table each represent the same meaning as in FIG. 2. 図２で表されるプライマーペアのSTING1 mRNAとの対応ならびに位置関係を表す。The correspondence and positional relationship between the primer pairs shown in Figure 2 and STING1 mRNA are shown. 図３で表されるプライマーペアのゲノムDNAとの対応ならびに位置関係を表す。3 shows the correspondence and positional relationship between the primer pairs shown in FIG. 3 and genomic DNA.
　本発明は、主に、ヒトSTING1遺伝子における、rs7380824、rs1131769、rs78233829およびrs11554776によって各々特定されるSNPの組み合わせに基づくハプロタイプを決定するための解析法に関するものであり、以下の構成を含む。すなわち、
（ａ）ヒトの生体試料からゲノムDNAおよびRNAを各々抽出する工程、
（ｂ）前記（ａ）記載の工程において抽出されたRNAから、逆転写反応（以下、「RT反応」（Reverse Transcription reaction）と略記することがある。）により、相補DNA（以下、「cDNA」（complementary DNA）と略記することがある。）を調製する工程、
（ｃ）（ｉ）前記（ｂ）記載の工程において調製されたcDNA、および（ｉｉ）当該４個のSNP（rs7380824、rs1131769、rs78233829およびrs11554776）のうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアを用いて、当該プライマーペアごとに別々に、所定の反応条件にてPCR（Polymerase Chain Reaction）を行う工程、
（ｄ）前記（ｃ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、
（ｅ）（ｉ）前記（ａ）記載の工程において抽出されたゲノムDNA、および（ｉｉ）当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを用いて、当該プライマーペアごとに別々に、所定の反応条件にてPCRを行う工程、
（ｆ）前記（ｅ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、ならびに
（ｇ）（ｉ）前記（ｄ）記載の工程において取得されたDNA配列情報から、ヒトSTING1遺伝子の一以上の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出し、（ｉｉ）前記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出し、（ｉｉｉ）前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程において抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の対立遺伝子のハプロタイプを決定する工程を含む。当該解析法の概要を図１に示す。 The present invention mainly relates to an analysis method for determining a haplotype based on a combination of SNPs each specified by rs7380824, rs1131769, rs78233829, and rs11554776 in the human STING1 gene, and includes the following configuration. That is,
(a) Extracting each of genomic DNA and RNA from a human biological sample;
(b) From the RNA extracted in the step described in (a) above, complementary DNA (hereinafter referred to as "cDNA") is generated by a reverse transcription reaction (hereinafter sometimes abbreviated as "RT reaction"). (sometimes abbreviated as complementary DNA).
(c) (i) cDNA prepared in the step described in (b) above, and (ii) at least three consecutive SNPs among the four SNPs (rs7380824, rs1131769, rs78233829 and rs11554776). A step of performing PCR (Polymerase Chain Reaction) separately under predetermined reaction conditions for each primer pair using one or more primer pairs designed to sandwich the
(d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof;
(e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, A step of separately performing PCR under predetermined reaction conditions;
(f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above. From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. Complementing the information includes determining the haplotypes of both alleles of the human STING1 gene. An overview of the analysis method is shown in Figure 1.
　ここで、本発明にかかるヒトSTING1遺伝子は、National Center for Biotechnology Information (NCBI) Reference Sequence: NP_938023.1（配列番号３７）にて特定されるヒトSTINGまたはそのアイソフォームをコードする遺伝子であり、NCBI Entrez Gene: 340061にて特定される。なお、本明細書において、「ヒトSTING」または「STING」と表記する場合、ヒトSTINGタンパク質のことを意味する。 Here, the human STING1 gene according to the present invention is a gene encoding human STING or its isoform specified by National Center for Biotechnology Information (NCBI) Reference Sequence: NP_938023.1 (SEQ ID NO: 37), and NCBI Identified by Entrez Gene: 340061. In addition, in this specification, when it is written as "human STING" or "STING", it means human STING protein.
　一塩基多型とは、「SNP」（Single Nucleotide Polymorphism）とも呼ばれ、ゲノムDNAの塩基配列の一塩基置換によって生じる多型を意味する。 Single nucleotide polymorphism, also called "SNP" ( Single Nucleotide Polymorphism ), refers to a polymorphism caused by a single nucleotide substitution in the base sequence of genomic DNA.
　本発明にかかるrs7380824は、ヒトの第５染色体の139477397番目に座位する塩基におけるシトシンおよびチミンの多型（相補鎖ゲノムDNA上では各々グアニンおよびアデニンの多型に相当する）を表し、当該塩基がシトシンである場合、NP_938023.1（配列番号３７）にて特定されるヒトSTINGの293番目のアミノ酸は野生型であるアルギニンとなり、当該塩基がチミンである場合、同アミノ酸はグルタミンとなり、このアミノ酸の多型を「R293Q」と表す。なお、rs7380824などのrs番号は、各SNPを特定するNCBIのdbSNPデータベースでの登録番号を表す。 rs7380824 according to the present invention represents a cytosine and thymine polymorphism (corresponding to guanine and adenine polymorphisms, respectively, on complementary strand genomic DNA) at the 139477397th base on human chromosome 5, and the base is If it is cytosine, the 293rd amino acid of human STING specified by NP_938023.1 (SEQ ID NO: 37) will be the wild type arginine, and if the base is thymine, the same amino acid will be glutamine, and this amino acid The polymorphism is expressed as "R293Q". Note that rs numbers such as rs7380824 represent registration numbers in NCBI's dbSNP database that specify each SNP.
　本発明にかかるrs1131769は、ヒトの第５染色体の139478334番目に座位する塩基におけるシトシンおよびチミンの多型（相補鎖ゲノムDNA上では各々グアニンおよびアデニンの多型に相当する）を表し、当該塩基がシトシンである場合、当該ヒトSTINGの232番目のアミノ酸は野生型であるアルギニンとなり、当該塩基がチミンである場合、同アミノ酸はヒスチジンとなり、このアミノ酸の多型を「R232H」と表す。 rs1131769 according to the present invention represents a cytosine and thymine polymorphism (corresponding to guanine and adenine polymorphisms, respectively, on complementary strand genomic DNA) at the 139478334th base on human chromosome 5, and the base is When the base is cytosine, the 232nd amino acid of the human STING is wild-type arginine; when the base is thymine, the amino acid is histidine, and the polymorphism of this amino acid is expressed as "R232H".
　本発明にかかるrs78233829は、ヒトの第５染色体の139478340番目に座位する塩基におけるシトシンおよびグアニンの多型（相補鎖ゲノムDNA上では各々グアニンおよびシトシンの多型に相当する）を表し、当該塩基がシトシンである場合、当該ヒトSTINGの230番目のアミノ酸は野生型であるグリシンとなり、当該塩基がグアニンである場合、同アミノ酸はアラニンとなり、このアミノ酸の多型を「G230A」と表す。なお、当該SNPは、上記のrs1131769のSNPと隣接している。 rs78233829 according to the present invention represents a cytosine and guanine polymorphism (corresponding to guanine and cytosine polymorphisms, respectively, on complementary strand genomic DNA) at the 139478340th base of human chromosome 5, and the base is When the base is cytosine, the 230th amino acid of the human STING is the wild type glycine, and when the base is guanine, the amino acid is alanine, and the polymorphism of this amino acid is designated as "G230A". Note that this SNP is adjacent to the SNP of rs1131769 described above.
　本発明にかかるrs11554776は、ヒトの第５染色体の139481493番目に座位する塩基におけるシトシンおよびチミンの多型（相補鎖ゲノムDNA上では各々グアニンおよびアデニンの多型に相当する）を表し、当該塩基がシトシンである場合、野生型であり、当該ヒトSTINGの71番目のアミノ酸はアルギニンとなり、当該塩基がチミンである場合、同アミノ酸はヒスチジンとなり、このアミノ酸の多型を「R71H」と表す。 rs11554776 according to the present invention represents a cytosine and thymine polymorphism (corresponding to guanine and adenine polymorphisms, respectively, on complementary strand genomic DNA) at the 139481493rd base on human chromosome 5, and the base is When the base is cytosine, it is wild type, and the 71st amino acid of the human STING is arginine; when the base is thymine, the amino acid is histidine, and the polymorphism of this amino acid is expressed as "R71H".
　ハプロタイプとは、一つの対立遺伝子に存在する遺伝子多型の組み合わせを言う。本発明におけるヒトSTING1遺伝子の４個のSNPの組み合わせに基づくハプロタイプのうち、表１に示されるハプロタイプについて各々略称が付されている。なお、「対立遺伝子」とは、二つの相同な遺伝子を構成する一方の遺伝子を意味し、「アレル」とも呼ばれる。 Haplotype refers to a combination of genetic polymorphisms present in one allele. Among the haplotypes based on the combination of four SNPs of the human STING1 gene in the present invention, the haplotypes shown in Table 1 are each given an abbreviation. Note that the term "allele" refers to one gene that constitutes two homologous genes, and is also called an "allele."
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　なお、表１中、「ハプロタイプ名」と記載された欄の垂直方向の欄の記載が、各ハプロタイプの略称を表す。それらの欄の水平方向に記載のアルファベット一文字は、４個のSNPを有するコドンに各々対応するアミノ酸の略号を表す。なお、このようなハプロタイプを決定することをハプロタイピングと言う。 In Table 1, the entries in the vertical column of the column labeled "Haplotype Name" represent the abbreviation of each haplotype. Each letter of the alphabet written horizontally in these columns represents the abbreviation of the amino acid corresponding to each codon having the four SNPs. Note that determining such haplotypes is called haplotyping.
　染色体は、原則として、二つの対立遺伝子の組み合わせからなる相同遺伝子を有するため、個体が有するハプロタイプは各対立遺伝子に由来する一組からなり、本発明におけるヒトSTING1遺伝子の４個のSNPの組み合わせに基づくハプロタイプには、例えば、表２に示される組み合わせが存在する。 Chromosomes, in principle, have homologous genes consisting of a combination of two alleles, so the haplotype that an individual has consists of one set derived from each allele. Based on the haplotypes, there are, for example, the combinations shown in Table 2.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　なお、表２中の右欄最下段の「RAHR」は、ヒトSTINGの71番目がアルギニン、230番目がアラニン、232番目がヒスチジンおよび293番目がアルギニンとなるハプロタイプを表す。 In addition, "RAHR" at the bottom of the right column in Table 2 represents a haplotype in which the 71st position of human STING is arginine, the 230th position is alanine, the 232nd position is histidine, and the 293rd position is arginine.
　本発明における「生体試料」とは、被検者から採取された何れの組織の一部でもよいが、採取およびそこからのDNAおよびRNAの抽出が比較的容易な血液、特に、有核細胞を含む全血が好ましい。血液試料は、採血時に使用される抗凝固剤（例えば、クエン酸ナトリウム等）が添加されていてもよく、EDTA採血管で採血されたものであってもよい。また、血液試料は、低温保存あるいは冷凍保存されていたものでもよい。 The "biological sample" in the present invention may be a part of any tissue collected from a subject, but blood, in particular nucleated cells, from which collection and extraction of DNA and RNA are relatively easy. Whole blood containing blood is preferred. The blood sample may be added with an anticoagulant (eg, sodium citrate, etc.) used during blood collection, or may be blood sampled using an EDTA blood collection tube. Further, the blood sample may be one that has been stored at low temperature or frozen.
　生体試料からのゲノムDNAの抽出は、当業者において公知の方法で実施でき、例えば、熱抽出法、アルカリ熱抽出法、フェノール・クロロホルム抽出法、スピンカラム法、磁性ビーズ法等を利用する方法、または市販のDNA抽出キット（例えば、NucleoSpin（登録商標）RNA BloodおよびNucleoSpin（登録商標）RNA/DNA Buffer Set（MACHEREY-NAGEL社）等）を用いる方法がある。 Extraction of genomic DNA from biological samples can be carried out by methods known to those skilled in the art, such as methods using thermal extraction, alkaline thermal extraction, phenol/chloroform extraction, spin column methods, magnetic bead methods, etc. Alternatively, there is a method using a commercially available DNA extraction kit (for example, NucleoSpin (registered trademark) RNA Blood and NucleoSpin (registered trademark) RNA/DNA Buffer Set (MACHEREY-NAGEL), etc.).
　生体試料からのRNAの抽出は、当業者において公知の方法で実施でき、例えば、AGPC法、オリゴdTカラムを用いる方法、または市販のRNA抽出キット（例えば、NucleoSpin（登録商標）RNA Blood（MACHEREY-NAGEL社）およびRNeasyカラム（Qiagen社）等）を用いる方法がある。 Extraction of RNA from biological samples can be performed by methods known to those skilled in the art, such as the AGPC method, a method using an oligo dT column, or a commercially available RNA extraction kit (for example, NucleoSpin® RNA Blood (MACHEREY- There are methods using RNeasy columns (NAGEL) and RNeasy columns (Qiagen, etc.).
　RT反応とは、逆転写酵素（例えば、PrimeScript（登録商標）RTase）によって、mRNA（messenger RNA）からcDNAを合成する反応を意味する。ここで、cDNAは、mRNAを鋳型として逆転写酵素によって生成され、mRNAと相補的な塩基配列を持つDNAである。RT反応は、オリゴdTプライマーをmRNAにアニーリングさせる反応、mRNAを鋳型としてオリゴdTプライマーを伸長させる逆転写反応および高温により逆転写酵素を失活させる反応からなり、当業者において公知の方法で実施でき、例えば、市販のキット（例えば、PrimeScript RT-PCR kit（タカラバイオ）等）を用いる方法がある。 The RT reaction refers to a reaction in which cDNA is synthesized from mRNA (messenger RNA) using a reverse transcriptase (eg, PrimeScript (registered trademark) RTase). Here, cDNA is DNA that is produced by reverse transcriptase using mRNA as a template and has a base sequence complementary to mRNA. The RT reaction consists of a reaction in which an oligo dT primer is annealed to mRNA, a reverse transcription reaction in which the oligo dT primer is extended using mRNA as a template, and a reaction in which reverse transcriptase is inactivated by high temperature, and can be carried out by methods known to those skilled in the art. For example, there is a method using a commercially available kit (eg, PrimeScript RT-PCR kit (Takara Bio), etc.).
　PCR（Polymerase Chain Reaction）とは、目的とするDNA領域を挟む２種のプライマーを用いて、当該領域のDNA断片を増幅する反応である。当該反応は、（ｉ）標的とするDNAを変性させ、（ｉｉ）プライマーをそのプライマー結合部位にアニーリングさせ、（ｉｉｉ）デオキシヌクレオシド三リン酸（dNTP）の存在下で、耐熱性DNAポリメラーゼ（例えば、Taq DNAポリメラーゼ等）によってプライマーを伸長させる一連の反応を複数回繰り返して実施できる。通常、当該反応は、サーマルサイクラーを用いて、最適化された温度、継続時間およびサイクル数にて行い、当該条件は、例えば、McPherson et al, PCR 2: A Practical Approach（July 27, 1995 by Oxford University）を参考に事前に設定することができる。例えば、Taq DNAポリメラーゼを使用するPCRでは、二本鎖DNAを、90℃以上の温度にて変性させ、プライマーを50～75℃の温度にてアニーリングさせ、プライマーを68～78℃の温度にて伸長させ、実施できる。 PCR (Polymerase Chain Reaction) is a reaction that uses two types of primers that sandwich a DNA region of interest to amplify a DNA fragment in that region. The reaction (i) denatures the targeted DNA, (ii) anneals the primer to its primer binding site, and (iii) injects a thermostable DNA polymerase (e.g. , Taq DNA polymerase, etc.) can be repeated multiple times. Typically, the reaction is performed using a thermal cycler at an optimized temperature, duration, and number of cycles; the conditions are, for example, as described by McPherson et al, PCR 2: A Practical Approach (July 27, 1995 by Oxford). It can be set in advance by referring to the University. For example, in PCR using Taq DNA polymerase, double-stranded DNA is denatured at temperatures above 90°C, primers are annealed at temperatures of 50-75°C, and primers are annealed at temperatures of 68-78°C. It can be extended and implemented.
　プライマーとは、本発明の解析法において実施されるPCRにおいて、増幅したいDNA領域（ターゲット領域）をForward方向とReverse方向から挟むように設計され、その領域の末端付近の塩基配列に相同な連続した十数～数十塩基長からなる一本鎖DNAであり、当業者において公知の方法、例えば、ホスホロアミダイト法等で合成することができる。本明細書において、ターゲット領域を挟むように設計された、Forward方向とReverse方向の一組のプライマーの組み合わせを「プライマーペア」と言い、プライマーの設計は、隣接する領域を含むターゲット領域の塩基配列情報を用いて、既存のプライマー設計ソフトウェア、例えば、OLIGO（登録商標）Primer Analysis Software（Molecular Biology Insights社）、Primer3（https://primer3.ut.ee/）またはPrimer-BLAST（https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi）等を用いて設計することができる。 Primers are designed to sandwich the DNA region to be amplified (target region) in the forward and reverse directions in the PCR performed in the analysis method of the present invention, and are continuous primers that are homologous to the base sequence near the end of the region. It is a single-stranded DNA having a length of ten to several dozen bases, and can be synthesized by methods known to those skilled in the art, such as the phosphoramidite method. In this specification, a combination of forward and reverse primers designed to sandwich a target region is referred to as a "primer pair", and the primer design is based on the base sequence of the target region including the adjacent region. The information can be used with existing primer design software, such as OLIGO® Primer Analysis Software (Molecular Biology Insights), Primer3 (https://primer3.ut.ee/) or Primer-BLAST (https:// It can be designed using tools such as www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi).
　PCRアンプリコンとは、PCRによって増幅したDNAのことを言う。 PCR amplicon refers to DNA amplified by PCR.
　本発明におけるシーケンシングまたはシーケンスとは、DNAの塩基配列を決定する方法であり、当業者において公知の方法で実施でき、例えば、従来から実施されているサンガーシーケンシング（ジデオキシシーケンシング）（Proc Natl Acad Sci U S A. 1977 Dec; 74(12): 5463-5467）やマクサム・ギルバートシーケンシング（Proc Natl Acad Sci U S A. 1977 February; 74(2): 560-564）等が挙げられる。さらに、次世代シーケンシング（NGS：Next Generation Sequencing）、例えば、パイロシーケンシング（Pyrosequencing）（GS Junior（登録商標）、GS Junior（登録商標）PlusまたはGS FLX+（登録商標）システム（GS FLX Titanium XL+（登録商標）およびGS FLX Titanium XLR70（登録商標））（Roche社）を用いた手法）、合成によるシーケンシング（SBS：Sequencing by Synthesis）（MiSeq（登録商標）、NextSeq（登録商標）500、HiSeq（登録商標）2500、HiSeq（登録商標）3000またはHiSeq（登録商標）4000（Illumina社）を用いた手法）、Ion Torrent（登録商標）半導体シーケンシング（Ion Torrent semiconductor sequencing）（Ion Proton（登録商標）System、Ion GeneStudio（登録商標）S5 SystemまたはIon Torrent（登録商標）Genexus（登録商標）System（Thermo Fisher SCENTIFIC社）を用いた手法）、ライゲーションによるシーケンシング（SBL：Sequencing by Ligation）（Genetic Analyzer V2.0（Thermo Fisher SCENTIFIC社）を用いたシーケンス手法）などが挙げられる。 Sequencing or sequencing in the present invention refers to a method for determining the base sequence of DNA, and can be carried out by methods known to those skilled in the art, such as Sanger sequencing (dideoxy sequencing) (Proc Natl. Acad Sci U S A. 1977 Dec; 74(12): 5463-5467) and Maxam-Gilbert sequencing (Proc Natl Acad Sci U S A. 1977 February; 74(2): 560-564). Furthermore, next generation sequencing ( NGS ), e.g. Pyrosequencing (GS Junior®, GS Junior® Plus or GS FLX+® systems (GS Sequencing by Synthesis (SBS) (MiSeq (registered trademark), NextSeq (registered trademark)) Ion Torrent semiconductor sequencing (Ion Proton) (registered trademark) System, Ion GeneStudio (registered trademark) S5 System or Ion Torrent (registered trademark) Genexus (registered trademark) System (Thermo Fisher SCENTIFIC)), Sequencing by Ligation (SBL) (sequencing method using Genetic Analyzer V2.0 (Thermo Fisher SCENTIFIC)).
　本発明にかかるSNPのジェノタイプとは、二つの対立遺伝子間の特定のSNPにおける塩基の組み合わせを言う。例えば、rs7380824にて特定されるSNPのジェノタイプは、二つの対立遺伝子上の同座位の塩基がともにシトシンである場合、「CC」で表され、シトシンおよびチミンである場合、「CT」で表され、両方がともにチミンである場合、「TT」で表される。また、rs1131769にて特定されるSNPのジェノタイプには、「CC」、「CT」および「TT」があり、rs78233829にて特定されるSNPのジェノタイプには、「CC」、「CG」および「GG」があり、rs11554776にて特定されるSNPのジェノタイプには、「CC」、「CT」および「TT」がある。なお、このようなジェノタイプを決定することをジェノタイピングと言う。 The SNP genotype according to the present invention refers to the combination of bases in a specific SNP between two alleles. For example, the genotype of the SNP specified in rs7380824 is expressed as “CC” if the bases at the same locus on two alleles are both cytosine, and “CT” if they are cytosine and thymine. and both are thymine, it is expressed as “TT”. Furthermore, the genotypes of the SNP identified by rs1131769 include "CC", "CT", and "TT", and the genotypes of the SNP identified by rs78233829 include "CC", "CG", and There is "GG", and the SNP genotypes specified at rs11554776 include "CC", "CT", and "TT". Note that determining such a genotype is called genotyping.
　本明細書において用いられる「約」とは、当業者において実施可能な範囲において、表記される数値を10％以内の範囲で下回って、または上回って変化してよいことを意味する。また、本明細書における範囲を示す表記「～」がある場合は、上限と下限を含有するものとする。 The term "about" as used herein means that it may vary by up to 10% below or above the stated numerical value within the range that is practicable by those skilled in the art. Further, in the present specification, when there is a notation "~" indicating a range, it includes an upper limit and a lower limit.
　本発明のハプロタイプ解析法において、上記（ｂ）記載の工程におけるRT反応は、例えば、アニーリング反応（RNA抽出物を65℃で５分間、その後、低温(例えば、４℃)で保持する）ののち、42℃で30分間、続いて、70℃で15分間、その後、4℃で保持することで実施できる。 In the haplotype analysis method of the present invention, the RT reaction in the step (b) above is performed, for example, after an annealing reaction (RNA extract is held at 65°C for 5 minutes and then held at a low temperature (e.g., 4°C)). , for 30 minutes at 42°C, followed by 15 minutes at 70°C, and then held at 4°C.
　本発明のハプロタイプ解析法において、上記（ｃ）（ｉｉ）記載の工程における、当該４個のSNPのうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアとしては、例えば、
（ｃ１）　当該４個のSNPすべてを挟むプライマーペア（例えば、（ｉ）配列番号１および２、（ｉｉ）配列番号３および４、ならびに（ｉｉｉ）配列番号５および６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）、
（ｃ２）　rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペア（例えば、（ｉ）配列番号７および８、（ｉｉ）配列番号９および１０、ならびに（ｉｉｉ）配列番号１１および１２から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）、ならびに
（ｃ３）　rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペア（例えば、（ｉ）配列番号１３および１４、（ｉｉ）配列番号１５および１６、ならびに（ｉｉｉ）配列番号１７および１８から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）が挙げられ（図２）、
前記（ｃ１）～（ｃ３）の各プライマーペアとして好ましくは、各々、配列番号１および２の一組の塩基配列からなる一本鎖DNAの組み合わせ、配列番号７および８の一組の塩基配列からなる一本鎖DNAの組み合わせ、ならびに配列番号１３および１４の一組の塩基配列からなる一本鎖DNAの組み合わせである。 In the haplotype analysis method of the present invention, one or more pairs of primers designed to sandwich at least three consecutive SNPs among the four SNPs in the step described in (c)(ii) above. For example, as a pair,
(c1) A primer pair sandwiching all the four SNPs (for example, a set of primers selected from (i) SEQ ID NO: 1 and 2, (ii) SEQ ID NO: 3 and 4, and (iii) SEQ ID NO: 5 and 6). a combination of single-stranded DNA consisting of base sequences),
(c2) Primer pair sandwiching the SNPs of rs7380824, rs1131769, and rs78233829 (for example, a pair selected from (i) SEQ ID NO: 7 and 8, (ii) SEQ ID NO: 9 and 10, and (iii) SEQ ID NO: 11 and 12) A combination of single-stranded DNA consisting of the nucleotide sequence of (iii) a combination of single-stranded DNA consisting of a set of base sequences selected from SEQ ID NO: 17 and 18) (Figure 2),
Each of the primer pairs (c1) to (c3) is preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NO: 1 and 2, and a pair of base sequences of SEQ ID NO: 7 and 8, respectively. and a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 13 and 14.
　本発明のハプロタイプ解析法において、上記（ｃ）記載の工程におけるPCRは、例えば、表３に示される条件で実施することができるが、プライマーとのアニーリング工程については、約55～約65℃にて約15～60秒間保持する条件で実施することもできる。 In the haplotype analysis method of the present invention, PCR in the step described in (c) above can be carried out, for example, under the conditions shown in Table 3, but the annealing step with the primers is performed at about 55 to about 65°C. It can also be carried out under conditions of holding for about 15 to 60 seconds.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　本発明のハプロタイプ解析法において、上記（ｄ）記載の工程におけるシーケンシングは、例えば、次世代シーケンシングで実施でき、好ましくは、合成によるシーケンシング（例えば、MiSeq（登録商標）（Illumina社）を用いた手法）で実施できる。 In the haplotype analysis method of the present invention, the sequencing in the step (d) above can be performed, for example, by next-generation sequencing, and is preferably performed by synthetic sequencing (for example, MiSeq (registered trademark) (Illumina)). It can be carried out using the method used in this study.
　本発明のハプロタイプ解析法において、上記（ｅ）（ｉｉ）記載の工程における、当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアとしては、例えば、
（ｅ１）rs7380824のSNPを挟むプライマーペア（例えば、（ｉ）配列番号１９および２０、（ｉｉ）配列番号２１および２２、ならびに（ｉｉｉ）配列番号２３および２４から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）、
（ｅ２）rs1131769およびrs78233829の二つのSNPを挟むプライマーペア（例えば、（ｉ）配列番号２５および２６、（ｉｉ）配列番号２７および２８、ならびに（ｉｉｉ）配列番号２９および３０から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）、ならびに
（ｅ３）rs11554776のSNPを挟むプライマーペア（例えば、（ｉ）配列番号３１および３２、（ｉｉ）配列番号３３および３４、ならびに（ｉｉｉ）配列番号３５および３６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせ）が挙げられ（図３）、
前記（ｅ１）～（ｅ３）の各プライマーペアとして好ましくは、各々、配列番号２１および２２の一組の塩基配列からなる一本鎖DNAの組み合わせ、配列番号２７および２８の一組の塩基配列からなる一本鎖DNAの組み合わせ、ならびに配列番号３３および３４の一組の塩基配列からなる一本鎖DNAの組み合わせである。 In the haplotype analysis method of the present invention, the primer pair sandwiching one or more SNPs among the four SNPs in the steps (e) and (ii) above may be, for example,
(e1) A primer pair sandwiching the SNP of rs7380824 (for example, from a set of base sequences selected from (i) SEQ ID NOs: 19 and 20, (ii) SEQ ID NOs: 21 and 22, and (iii) SEQ ID NOs: 23 and 24) a combination of single-stranded DNA),
(e2) A primer pair sandwiching the two SNPs rs1131769 and rs78233829 (for example, a pair selected from (i) SEQ ID NOs: 25 and 26, (ii) SEQ ID NOs: 27 and 28, and (iii) SEQ ID NOs: 29 and 30) A combination of single-stranded DNA consisting of the nucleotide sequence of A combination of single-stranded DNA consisting of a set of base sequences selected from numbers 35 and 36) (Fig.
Each of the primer pairs (e1) to (e3) is preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 21 and 22, and a pair of base sequences of SEQ ID NOs: 27 and 28, respectively. and a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 33 and 34.
　本発明のハプロタイプ解析法において、上記（ｅ）記載の工程におけるPCRは、例えば、表３に示される条件と同じ条件で実施することができるが、プライマーとのアニーリング工程については、55～65℃にて約15～60秒間保持する条件で実施することもできる。 In the haplotype analysis method of the present invention, PCR in the step described in (e) above can be carried out under the same conditions as shown in Table 3, but the annealing step with the primers can be carried out at 55 to 65°C. It can also be carried out under conditions of holding for about 15 to 60 seconds.
　本発明のハプロタイプ解析法において、上記（ｆ）記載の工程におけるシーケンシングは、例えば、サンガーシーケンシングで実施できる。 In the haplotype analysis method of the present invention, the sequencing in the step (f) above can be performed, for example, by Sanger sequencing.
　本発明のハプロタイプ解析法において、上記（ｇ）（ｉ）記載の工程における、上記（ｄ）記載の工程において取得したDNA配列情報から、ヒトSTING1遺伝子の両方の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出する方法としては、例えば、以下の方法が挙げられる。 In the haplotype analysis method of the present invention, from the DNA sequence information obtained in the step described in (d) above in the step described in (g) (i) above, the four SNPs are determined for both alleles of the human STING1 gene. Examples of methods for extracting haplotypes based on combinations include the following methods.
　すなわち、まず、Miseq（登録商標）Systemによって取得したDNA塩基配列情報を、ヒトゲノム参照配列にマッピングし、FreeBayes（version 1.3.5, GitHub, https://github.com/freebayes/freebayes）を用いてハプロタイプの変異検出解析を実施する。同解析には、測定SNPがQV40以上の塩基を使用し、ヘテロコールの閾値は、Allele frequency > 0.1とする。出力されたVCFファイルについてVcflib（version 1.0.2, GitHub, https://github.com/vcflib/vcflib）を使用し、マルチアレルを単一のコールに分解して、VCFtools（version 0.1.16, GitHub, https://github.com/vcftools/vcftools）を用いてコールされたポジションを測定SNPのみに絞り込み、また、マルチアレルでコールされた場合は、bcftools（version 1.12, GitHub, https://github.com/samtools/bcftools）で個々のアレルに分解する。最終的に、WhatsHap（version 1.0, GitHub, https://github.com/whatshap/whatshap/）を使用して検出された測定SNPsのハプロタイプフェージングを行い、bcftoolsを用いて個別のvcfファイルをマージして、ハプロタイプの結果を取得する。 That is, first, the DNA base sequence information obtained by Miseq (registered trademark) System was mapped to the human genome reference sequence, and then mapped using FreeBayes (version 1.3.5, GitHub, https://github.com/freebayes/freebayes). Perform haplotype mutation detection analysis. For this analysis, bases with measured SNPs of QV40 or higher are used, and the heterocall threshold is Allele frequency > 0.1. For the output VCF file, use Vcflib (version 1.0.2, GitHub, https://github.com/vcflib/vcflib) to decompose multi-alleles into a single call, and use VCFtools (version 0.1.16, GitHub, https://github.com/vcftools/vcftools) is used to narrow down the called positions to only measured SNPs, and if called for multi-alleles, use bcftools (version 1.12, GitHub, https:// github.com/samtools/bcftools) to break it down into individual alleles. Finally, we performed haplotype phasing of the detected measured SNPs using WhatsHap (version 1.0, GitHub, https://github.com/whatshap/whatshap/) and merged the separate vcf files using bcftools. to obtain haplotype results.
　本発明のハプロタイプ解析法において、上記（ｇ）（ｉｉ）記載の工程における、上記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出する方法としては、例えば、以下の方法が挙げられる。 In the haplotype analysis method of the present invention, a method for extracting the genotype at each of the four SNPs from the DNA sequence information obtained in the step (f) above in the step (g) (ii) above. For example, the following method may be mentioned.
　すなわち、まず、ab1ファイルを、トリミングしたデータ塩基配列情報およびクオリティスコアをfastq形式で出力し、SeqKit（version 2.1.0, GitHub, https://github.com/shenwei356/seqkit）を用いてfastq形式からfasta形式ファイルに変換する。次に、測定遣伝子領域の配列とアライメントし、相同性および塩基配列データにおいてQV値が30以上である塩基割合が95%以上であることが確認できたファイルより、Chromas（version 2.6.6, Technelysium, http://technelysium.com.au/wp/chromas/）を用いて、各SNPのジェノタイプを検出する。 That is, first, output the trimmed data nucleotide sequence information and quality score of the ab1 file in fastq format, and convert it to fastq format using SeqKit (version 2.1.0, GitHub, https://github.com/shenwei356/seqkit). Convert from to fasta format file. Next, we aligned it with the sequence of the measured gene region, and from the file in which it was confirmed that the percentage of bases with a QV value of 30 or more in homology and base sequence data was 95% or more, Chromas (version 2.6.6 , Technelysium, http://technelysium.com.au/wp/chromas/) to detect the genotype of each SNP.
　二つの対立遺伝子間では、その遺伝子発現量が極端に相違する場合がある。そのため、そのようなケースでは、本発明の解析法のように、生体試料から抽出されたRNAに基づいてハプロタイピングを実施する場合、片方の対立遺伝子におけるSNPのハプロタイプしか決定できない場合がある。そのような場合には、別途取得した各SNPにおけるジェノタイプに関する情報を、当該ハプロタイプに関する情報に加えることで、一方の対立遺伝子におけるSNPのハプロタイプを導き出すことができる。しかしながら、本発明の解析法においては、二つの対立遺伝子におけるSNPのハプロタイプに関する情報が取得可能な場合においても、その精度を保証するため、各SNPにおけるジェノタイプに関する情報で補完することで、当該４個のSNPの組み合わせに基づくハプロタイプを決定する方法を選択した。 There are cases where the gene expression levels between two alleles are extremely different. Therefore, in such cases, when performing haplotyping based on RNA extracted from a biological sample, as in the analysis method of the present invention, it may be possible to determine only the haplotype of the SNP in one allele. In such a case, the haplotype of the SNP in one allele can be derived by adding separately acquired information regarding the genotype for each SNP to the information regarding the haplotype. However, in the analysis method of the present invention, even when information on the haplotype of a SNP in two alleles can be obtained, in order to guarantee its accuracy, it is supplemented with information on the genotype of each SNP, so that the four We chose a method to determine haplotypes based on combinations of SNPs.
　本発明のハプロタイプ解析法において、上記（ｇ）（ｉｉｉ）記載の工程における、前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程にて抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の当該対立遺伝子のハプロタイプを決定する方法としては、例えば、以下の方法が挙げられる。
（α）　ハプロタイプが２種類確認され、ジェノタイプの結果と符合する場合は、得られたハプロタイプを最終的な結果として決定する。
（β）　ハプロタイプが１種類確認され、ジェノタイプがヘテロ型の場合は、ジェノタイピングで得られた２個のアレルから、ハプロタイピングで得られた１個のアレルを引き去ることで、一方のハプロタイプを確定する。
（γ）　ジェノタイプが全領域でホモ体であり、ハプロタイプでも同じ型が得られた場合は、得られた型のホモ接合型と決定する。
（δ）　ジェノタイピングの結果と、ハプロタイピングの結果に矛盾がある場合は、遺伝型は未確定とする。 In the haplotype analysis method of the present invention, in the step (g) (iii) above, the information regarding the haplotype extracted in the step (i) above is converted to the genotype extracted in the step (ii) above. Examples of methods for determining the haplotypes of both alleles of the human STING1 gene by supplementing with information on the STING1 gene include the following method.
(α) If two haplotypes are confirmed and match the genotype results, the obtained haplotype is determined as the final result.
(β) If one haplotype is confirmed and the genotype is heterozygous, one haplotype can be created by subtracting one allele obtained by haplotyping from the two alleles obtained by genotyping. Confirm.
(γ) If the genotype is homozygous in the entire region and the same haplotype is obtained, it is determined to be homozygous for the obtained type.
(δ) If there is a discrepancy between the genotyping results and haplotyping results, the genotype is undetermined.
　本発明は、例えば、下記［１］～［１８］、［１－１］および［１－２］に挙げられる実施態様を各々提供する。
［１］　rs7380824、rs1131769、rs78233829およびrs11554776によって各々特定される、ヒトSTING1遺伝子における４個の一遺伝子多型(SNP)の組み合わせに基づくハプロタイプを決定するための方法であって、
（ａ）ヒトの生体試料からゲノムDNAおよびRNAを各々抽出する工程、
（ｂ）前記（ａ）記載の工程において抽出されたRNAから、RT反応により、cDNAを調製する工程、
（ｃ）（ｉ）前記（ｂ）記載の工程において調製されたcDNA、および（ｉｉ）当該４個のSNPのうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
（ｄ）前記（ｃ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、
（ｅ）（ｉ）前記（ａ）記載の工程において抽出されたゲノムDNA、および（ｉｉ）当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
（ｆ）前記（ｅ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、ならびに
（ｇ）（ｉ）前記（ｄ）記載の工程において取得されたDNA配列情報から、ヒトSTING1遺伝子の一以上の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出し、（ｉｉ）前記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出し、（ｉｉｉ）前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程において抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の対立遺伝子のハプロタイプを決定する工程を含む、当該方法；
［２］　ヒトの生体試料が全血である、前記［１］記載の方法；
［３］　前項［１］（ｃ）（ｉｉ）記載の一組以上のプライマーペアが、（ａ）当該４個のSNPすべてを挟むプライマーペア、（ｂ）rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペア、ならびに（ｃ）rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペアである、前記［１］または［２］記載の方法；
［４］　前項［３］記載の当該４個のSNPすべてを挟むプライマーペアが、
（ｉ）配列番号１および２、（ｉｉ）配列番号３および４、ならびに（ｉｉｉ）配列番号５および６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［３］記載の方法；
［５］　前項［３］（ｂ）記載のrs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペアが、（ｉ）配列番号７および８、（ｉｉ）配列番号９および１０、ならびに（ｉｉｉ）配列番号１１および１２から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［３］記載の方法；
［６］　前項［３］（ｃ）記載のrs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペアが、（ｉ）配列番号１３および１４、（ｉｉ）配列番号１５および１６、ならびに（ｉｉｉ）配列番号１７および１８から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［３］記載の方法；
［７］　前項［１］（ｂ）記載のRT反応が、抽出されたRNAを約65℃で約５分間、その後、約４℃で保持したのち、約42℃で約30分間、続いて、約70℃で約15分間、その後、約４℃で保持することで実施される、前記［１］～［６］の何れか一項記載の方法；
［８］　前項［１］（ｃ）記載のPCRが、上表３に示される条件で実施される、前記［１］～［７］の何れか一項記載の方法；
［９］　前項［１］（ｄ）記載のシーケンシングが、次世代シーケンシングで実施される、前記［１］～［８］の何れか一項記載の方法；
［１０］　次世代シーケンシングが、パイロシーケンシング、合成によるシーケンシング、Ion Torrent（登録商標）半導体シーケンシングまたはライゲーションによるシーケンシングである、前記［９］記載の方法；
［１１］　次世代シーケンシングが合成によるシーケンシングであり、当該合成によるシーケンシングが、MiSeq（登録商標）（Illumina社）を用いた手法で実施される、前記［９］記載の方法；
［１２］　前項［１］（ｅ）（ｉｉ）記載の４個のSNPのうちの一以上のSNPを各々挟むプライマーペアが、（ａ）rs7380824のSNPを挟むプライマーペア、（ｂ）rs1131769およびrs78233829の二つのSNPを挟むプライマーペアならびに（ｃ）rs11554776のSNPを挟むプライマーペアからなる、前記［１］～［１１］の何れか一項記載の方法；
［１３］　前項［１２］（ａ）記載のrs7380824のSNPを挟むプライマーペアが、（ｉ）配列番号１９および２０、（ｉｉ）配列番号２１および２２、ならびに（ｉｉｉ）配列番号２３および２４から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［１２］記載の方法；
［１４］前項［１２］（ｂ）記載のrs1131769およびrs78233829の二つのSNPを挟むプライマーペアが、（ｉ）配列番号２５および２６、（ｉｉ）配列番号２７および２８、ならびに（ｉｉｉ）配列番号２９および３０から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［１２］記載の方法；
［１５］前項［１２］（ｃ）記載のrs11554776のSNPを挟むプライマーペアが、（ｉ）配列番号３１および３２、（ｉｉ）配列番号３３および３４、ならびに（ｉｉｉ）配列番号３５および３６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、前記［１２］記載の方法；
［１６］　前項［１］（ｅ）記載のPCRが、上表３に示される条件で実施される、前記［１］～［１５］の何れか一項記載の方法；
［１７］　前項［１］（ｆ）記載のシーケンシングが、サンガーシーケンシングである、前記［１］～［１６］の何れか一項記載の方法；
［１８］　rs7380824、rs1131769、rs78233829およびrs11554776によって各々特定される、ヒトSTING1遺伝子における４個の一遺伝子多型(SNP)の組み合わせに基づくハプロタイプを決定するための方法であって、
（ａ）ヒトの生体試料からゲノムDNAおよびRNAを各々抽出する工程、
（ｂ）前記（ａ）記載の工程において抽出されたRNAから、RT反応により、cDNAを調製する工程、
（ｃ）（ｉ）前記（ｂ）記載の工程において調製されたcDNA、および（ｉｉ）当該４個のSNPのうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
（ｄ）前記（ｃ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、
（ｅ）（ｉ）前記（ａ）記載の工程において抽出されたゲノムDNA、および（ｉｉ）当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
（ｆ）前記（ｅ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、ならびに
（ｇ）（ｉ）前記（ｄ）記載の工程において取得されたDNA配列情報から、ヒトSTING1遺伝子の一以上の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出し、（ｉｉ）前記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出し、（ｉｉｉ）前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程において抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の対立遺伝子のハプロタイプを決定する工程からなり、
ここで、前記（ｂ）記載のRT反応が、上記に示される条件（抽出されたRNAを約65℃で約５分間、その後、約４℃で保持したのち、約42℃で約30分間、続いて、約70℃で約15分間、その後、約４℃で保持する）で実施され、
前記（ｃ）（ｉｉ）記載の一組以上のプライマーペアが、（ａ）当該４個のSNPすべてを挟むプライマーペア（好ましくは、配列番号１および２の一組の塩基配列からなる一本鎖DNAの組み合わせ）、（ｂ）rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペア（好ましくは、配列番号７および８の一組の塩基配列からなる一本鎖DNAの組み合わせ）、ならびに（ｃ）rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペア（好ましくは、配列番号１３および１４の一組の塩基配列からなる一本鎖DNAの組み合わせ）であり、
前記（ｃ）記載のPCRが、上表３に示される条件で実施され、
前記（ｄ）記載のシーケンシングが、次世代シーケンシング（好ましくは、合成によるシーケンシング、より好ましくは、MiSeq（登録商標）（Illumina社）を用いた手法）で実施され、
前記（ｅ）（ｉｉ）記載の４個のSNPのうちの一以上のSNPを各々挟むプライマーペアが、（ａ）rs7380824のSNPを挟むプライマーペア（好ましくは、配列番号２１および２２の一組の塩基配列からなる一本鎖DNAの組み合わせ）、（ｂ）rs1131769およびrs78233829の二つのSNPを挟むプライマーペア（好ましくは、配列番号２７および２８の一組の塩基配列からなる一本鎖DNAの組み合わせ）、ならびに（ｃ）rs11554776のSNPを挟むプライマーペア（好ましくは、配列番号３３および３４の一組の塩基配列からなる一本鎖DNAの組み合わせ）からなり、
前記（ｅ）記載のPCRが、上表３に示される条件で実施され、
前記（ｆ）記載のシーケンシングが、サンガーシーケンシングである、当該方法；
［１－１］　配列番号１～１８からなる群から選択される塩基配列からなる一本鎖DNA；ならびに
［１－２］　配列番号１９～３６からなる群から選択される塩基配列からなる一本鎖DNA。 The present invention provides, for example, each of the embodiments listed in [1] to [18], [1-1], and [1-2] below.
[1] A method for determining a haplotype based on a combination of four monogenic polymorphisms (SNPs) in the human STING1 gene, each identified by rs7380824, rs1131769, rs78233829 and rs11554776, comprising:
(a) Extracting each of genomic DNA and RNA from a human biological sample;
(b) a step of preparing cDNA by RT reaction from the RNA extracted in the step described in (a) above;
(c) (i) cDNA prepared in the step described in (b) above, and (ii) a set designed to sandwich at least three consecutive SNPs among the four SNPs. Using the above primer pairs, performing PCR separately for each primer pair,
(d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof;
(e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, The process of performing PCR separately,
(f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above. From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. The method comprises determining the haplotypes of both alleles of the human STING1 gene by supplementing the information;
[2] The method according to [1] above, wherein the human biological sample is whole blood;
[3] The one or more primer pairs described in the preceding paragraph [1] (c) (ii) are (a) primer pairs that sandwich all of the four SNPs, (b) primers that sandwich the SNPs of rs7380824, rs1131769, and rs78233829. and (c) the method according to [1] or [2] above, which is a primer pair sandwiching SNPs of rs1131769, rs78233829 and rs11554776;
[4] The primer pair sandwiching all four SNPs described in the previous section [3] is
The [3 ] The method described;
[5] The primer pair sandwiching the SNPs of rs7380824, rs1131769, and rs78233829 described in the previous section [3] (b) is (i) SEQ ID NO: 7 and 8, (ii) SEQ ID NO: 9 and 10, and (iii) SEQ ID NO: 11. and the method according to [3] above, which is a combination of single-stranded DNA consisting of a set of base sequences selected from 12;
[6] The primer pair sandwiching the SNPs of rs1131769, rs78233829, and rs11554776 described in the previous section [3] (c) is (i) SEQ ID NO: 13 and 14, (ii) SEQ ID NO: 15 and 16, and (iii) SEQ ID NO: 17. and the method according to [3] above, which is a combination of single-stranded DNA consisting of a set of base sequences selected from 18;
[7] The RT reaction described in the previous section [1] (b) is performed by subjecting the extracted RNA to about 65°C for about 5 minutes, then holding it at about 4°C, and then at about 42°C for about 30 minutes, The method according to any one of [1] to [6] above, which is carried out at about 70°C for about 15 minutes and then held at about 4°C;
[8] The method according to any one of [1] to [7] above, wherein the PCR described in [1](c) above is carried out under the conditions shown in Table 3 above;
[9] The method according to any one of [1] to [8] above, wherein the sequencing described in [1](d) above is performed by next-generation sequencing;
[10] The method according to [9] above, wherein the next generation sequencing is pyrosequencing, sequencing by synthesis, Ion Torrent (registered trademark) semiconductor sequencing, or sequencing by ligation;
[11] The method according to [9] above, wherein the next generation sequencing is synthetic sequencing, and the synthetic sequencing is performed using MiSeq (registered trademark) (Illumina);
[12] Primer pairs that sandwich one or more of the four SNPs described in [1] (e) and (ii) above are (a) a primer pair that sandwiches the SNP of rs7380824, (b) rs1131769 and rs78233829. The method according to any one of [1] to [11] above, comprising a primer pair sandwiching the two SNPs and (c) a primer pair sandwiching the SNP rs11554776;
[13] The primer pair sandwiching the SNP of rs7380824 described in [12] (a) above is selected from (i) SEQ ID NO: 19 and 20, (ii) SEQ ID NO: 21 and 22, and (iii) SEQ ID NO: 23 and 24. The method according to [12] above, which is a combination of single-stranded DNA consisting of a set of base sequences;
[14] The primer pair sandwiching the two SNPs rs1131769 and rs78233829 described in the previous section [12] (b) is (i) SEQ ID NO: 25 and 26, (ii) SEQ ID NO: 27 and 28, and (iii) SEQ ID NO: 29. and the method according to [12] above, which is a combination of single-stranded DNA consisting of a set of base sequences selected from 30;
[15] The primer pair sandwiching the SNP of rs11554776 described in the previous section [12] (c) is selected from (i) SEQ ID NO: 31 and 32, (ii) SEQ ID NO: 33 and 34, and (iii) SEQ ID NO: 35 and 36. The method according to [12] above, which is a combination of single-stranded DNA consisting of a set of base sequences;
[16] The method according to any one of [1] to [15] above, wherein the PCR described in [1] (e) above is carried out under the conditions shown in Table 3 above;
[17] The method according to any one of [1] to [16] above, wherein the sequencing described in [1](f) is Sanger sequencing;
[18] A method for determining a haplotype based on a combination of four monogenic polymorphisms (SNPs) in the human STING1 gene, each identified by rs7380824, rs1131769, rs78233829 and rs11554776, comprising:
(a) Extracting each of genomic DNA and RNA from a human biological sample;
(b) a step of preparing cDNA by RT reaction from the RNA extracted in the step described in (a) above;
(c) (i) cDNA prepared in the step described in (b) above, and (ii) a set designed to sandwich at least three consecutive SNPs among the four SNPs. Using the above primer pairs, performing PCR separately for each primer pair,
(d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof;
(e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, The process of performing PCR separately,
(f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above. From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. The process consists of determining the haplotypes of both alleles of the human STING1 gene by supplementing with information,
Here, the RT reaction described in (b) above is carried out under the conditions shown above (extracted RNA is kept at about 65°C for about 5 minutes, then held at about 4°C, then at about 42°C for about 30 minutes, followed by a temperature of about 70°C for about 15 minutes, followed by a hold at about 4°C),
The one or more primer pairs described in (c)(ii) above are (a) a primer pair sandwiching all four SNPs (preferably a single strand consisting of a pair of nucleotide sequences SEQ ID NOs: 1 and 2); (a combination of DNA), (b) a primer pair sandwiching SNPs of rs7380824, rs1131769 and rs78233829 (preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 7 and 8), and (c) rs1131769, A primer pair sandwiching the SNPs of rs78233829 and rs11554776 (preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 13 and 14),
The PCR described in (c) above is carried out under the conditions shown in Table 3 above,
The sequencing described in (d) above is performed by next generation sequencing (preferably synthetic sequencing, more preferably a method using MiSeq (registered trademark) (Illumina)),
The primer pair sandwiching one or more SNPs among the four SNPs described in (e) and (ii) above is (a) the primer pair sandwiching the rs7380824 SNP (preferably a pair of SEQ ID NOs: 21 and 22). (b) a primer pair sandwiching two SNPs, rs1131769 and rs78233829 (preferably, a combination of single-stranded DNA consisting of a pair of base sequences SEQ ID NOs: 27 and 28) , and (c) consisting of a primer pair (preferably a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 33 and 34) sandwiching the SNP of rs11554776,
The PCR described in (e) above is carried out under the conditions shown in Table 3 above,
The method, wherein the sequencing described in (f) above is Sanger sequencing;
[1-1] Single-stranded DNA consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 18; and [1-2] Single strand DNA consisting of a base sequence selected from the group consisting of SEQ ID NOS: 19 to 36 Strand DNA.
　本明細書において、明示的に引用されるすべての特許文献および非特許文献もしくは参考文献の内容は、全て本明細書の一部としてここに引用し得る。 In this specification, the contents of all patent documents and non-patent documents or references explicitly cited may be cited here as part of this specification.
　本発明を以下の実施例によってさらに詳しく説明するが、本発明の範囲はこれに限定されない。本発明の記載に基づき種々の変更、修飾が当業者には可能であり、これらの変更、修飾も本発明に含まれる。 The present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.
実施例１：血液試料からのゲノムDNA抽出
　前処理として、凍結状態の血液に血液と等量のLysis Buffer DL（NucleoSpin RNA/DNA Buffer Set（Macherey-Nagel社）に含まれる試薬）を添加し、融解および溶解した。当該等量混合液800μLを2.0mLチューブに分取した。Proteinase K溶液（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）10μLを添加し、室温（18～25℃）で15分間激しく振とうした。同反応液に70％エタノール 400μLを添加し、混合した。同混合液をNucleoSpin RNA Blood Column（NucleoSpin RNA/DNA Buffer Setに含まれるカラム）に添加して遠心し、核酸をシリカメンブレンに吸着させた。Buffer DNA Wash（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）500μLをカラムに通してメンブレンの洗浄を行った。再度、Buffer DNA Wash 500μLをカラムに通してメンブレンの洗浄を行い、乾燥させた。Buffer DNA Elute（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）100μLを加え、DNAを溶出させ、回収した。回収されたDNAは、-25～-15℃下で冷凍保存された。 Example 1: As a pretreatment for genomic DNA extraction from blood samples , an equal amount of Lysis Buffer DL (a reagent included in the NucleoSpin RNA/DNA Buffer Set (Macherey-Nagel)) was added to frozen blood, Melted and dissolved. 800 μL of the equal volume of the mixed solution was dispensed into a 2.0 mL tube. 10 μL of Proteinase K solution (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was added, and the mixture was shaken vigorously for 15 minutes at room temperature (18-25°C). 400 μL of 70% ethanol was added to the same reaction solution and mixed. The same mixture was added to a NucleoSpin RNA Blood Column (column included in the NucleoSpin RNA/DNA Buffer Set) and centrifuged to adsorb the nucleic acid onto the silica membrane. The membrane was washed by passing 500 μL of Buffer DNA Wash (a reagent included in the NucleoSpin RNA/DNA Buffer Set) through the column. Once again, 500 μL of Buffer DNA Wash was passed through the column to wash the membrane, and it was dried. 100 μL of Buffer DNA Elute (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was added to elute and collect the DNA. The recovered DNA was stored frozen at -25 to -15°C.
　抽出されたDNAの収量および品質を確認した。Qubit Fluorometer（Thermo Fisher Scientific社）を用いて蛍光定量を行い、Qubit濃度として、600ng以上（濃度10ng/μL以上）であることを確認した。 The yield and quality of the extracted DNA was confirmed. Fluorescence quantification was performed using a Qubit Fluorometer (Thermo Fisher Scientific), and the Qubit concentration was confirmed to be 600 ng or more (concentration 10 ng/μL or more).
実施例２：血液試料からのTotal RNA抽出
　実施例１と同様に前処理された血液/Lysis Buffer DL等量混合液800μLを2.0mLチューブに分取した。Proteinase K溶液10μLを添加し、室温（18～25℃）で15分間激しく振とうした。同反応液に70％エタノール 400μLを添加し、混合した。同混合液をNucleoSpin RNA Blood Columnに添加して遠心し、核酸をシリカメンブレンに吸着させた。Membrane Desalting Buffer（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）350μLをカラムに添加し、メンブレンの脱塩を行った。rDNase溶液（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）95μLをメンブレンの中央へ添加し、室温で15分間インキュベートした。Buffer RB2（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）200μLをカラムに通してメンブレンの洗浄を行った。続いて、Buffer RB3（NucleoSpin RNA/DNA Buffer Setに含まれる試薬）600μLをカラムに通してメンブレンの洗浄を行った。さらに、Buffer RB3 250μLをカラムに通してメンブレンの洗浄を行い、メンブレンを乾燥させた。大塚蒸留水 60μLをカラムに加え、RNAを溶出させ、回収した。回収されたRNAは、-85～-70℃下で冷凍保存された。 Example 2: Extraction of Total RNA from Blood Samples 800 μL of an equal volume mixture of pretreated blood/Lysis Buffer DL in the same manner as in Example 1 was dispensed into a 2.0 mL tube. 10 μL of Proteinase K solution was added, and the mixture was shaken vigorously for 15 minutes at room temperature (18-25°C). 400 μL of 70% ethanol was added to the same reaction solution and mixed. The same mixture was added to a NucleoSpin RNA Blood Column and centrifuged to adsorb the nucleic acid onto the silica membrane. 350 μL of Membrane Desalting Buffer (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was added to the column to desalt the membrane. 95 μL of rDNase solution (reagent included in NucleoSpin RNA/DNA Buffer Set) was added to the center of the membrane and incubated at room temperature for 15 minutes. The membrane was washed by passing 200 μL of Buffer RB2 (a reagent included in the NucleoSpin RNA/DNA Buffer Set) through the column. Subsequently, 600 μL of Buffer RB3 (a reagent included in the NucleoSpin RNA/DNA Buffer Set) was passed through the column to wash the membrane. Furthermore, the membrane was washed by passing 250 μL of Buffer RB3 through the column, and the membrane was dried. 60 μL of Otsuka distilled water was added to the column, and RNA was eluted and collected. The recovered RNA was stored frozen at -85 to -70°C.
　抽出されたTotal RNAの収量および品質を確認した。Agilent 4200 TapeStation（Agilent Technologies社）を用いて、濃度およびRINe値(RNA Integrity Number equivalent)を測定し、TapeStation濃度として、800ng以上（濃度：12.5ng/μL以上）、RINe値として7.0以上（参考値）であることを確認した。 The yield and quality of the extracted total RNA was confirmed. The concentration and RINe value (RNA Integrity Number equivalent) were measured using Agilent 4200 TapeStation (Agilent Technologies), and the TapeStation concentration was 800 ng or more (concentration: 12.5 ng/μL or more), and the RINe value was 7.0 or more (reference value). ).
実施例３：サンガーシークエンスのためのPCR産物の調製
　各SNPを挟む領域のPCR産物を各々調製した。 Example 3: Preparation of PCR products for Sanger sequencing PCR products of the region sandwiching each SNP were prepared.
　PCRに使用されたプライマーペアのうち、rs7380824のSNPを挟むプライマーペアとして、各々配列番号２１および２２からなるプライマー、rs1131769およびrs78233829の二つのSNPを挟むプライマーペアとして、各々配列番号２７および２８からなるプライマー、rs11554776のSNPを挟むプライマーペアとして、各々配列番号３３および３４からなるプライマーが各々使用された（図３）。なお、以下、各々配列番号２１および２２からなるプライマーにより挟まれる領域を「領域１」とし、各々配列番号２７および２８からなるプライマーにより挟まれる領域を「領域２」とし、各々配列番号３３および３４からなるプライマーにより挟まれる領域を「領域３」とする（図５参照）。 Among the primer pairs used for PCR, the primer pair that sandwiched the SNP of rs7380824 consisted of SEQ ID NOs: 21 and 22, and the primer pair that sandwiched the two SNPs of rs1131769 and rs78233829 consisted of SEQ ID NOs: 27 and 28, respectively. Primers consisting of SEQ ID NOs: 33 and 34 were used as primer pairs sandwiching the SNP of rs11554776 (FIG. 3). Hereinafter, the region sandwiched by the primers consisting of SEQ ID NOs: 21 and 22, respectively, will be referred to as "region 1," the area sandwiched by the primers consisting of SEQ ID NOs: 27 and 28, respectively, will be referred to as "region 2," and the region sandwiched by the primers consisting of SEQ ID NOs: 27 and 28, respectively, will be referred to as "region 2." The region sandwiched by the primers consisting of the following is referred to as "region 3" (see FIG. 5).
　実施例１で調製されたDNA溶液（DNA 100ng含有）に、2×Gflex PCR Buffer（Mg2+, dNTP plus）（タカラバイオ社）25μL、前記各プライマーペア溶液（forward primerおよびReverse primer）各10pmol相当量およびTks Gflex DNA Polymerase（1.25 units/μL）（タカラバイオ社）１μLを混合し、合計容量50μLとなるようNuclease Free水を添加し、混合して、PCR反応液を各々調製した。 To the DNA solution prepared in Example 1 (containing 100 ng of DNA), 25 μL of 2×Gflex PCR Buffer (Mg 2+ , dNTP plus) (Takara Bio Inc.) and 10 pmol each of the above primer pair solutions (forward primer and reverse primer) were added. The corresponding amount and 1 μL of Tks Gflex DNA Polymerase (1.25 units/μL) (Takara Bio Inc.) were mixed, Nuclease Free water was added to make the total volume 50 μL, and the mixture was mixed to prepare each PCR reaction solution.
　当該各PCR反応液を、Veriti 200 サーマルサイクラー（Thermo Fisher Scientific社）にセットし、表４に示されるPCR条件にて、PCRを実施した。 Each of the PCR reaction solutions was set in a Veriti 200 thermal cycler (Thermo Fisher Scientific), and PCR was performed under the PCR conditions shown in Table 4.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　当該各PCR産物を各々AMPure XP（Beckman Coulter社）により精製し、Agilent2200/4200 TapeStationを用いて、濃度およびピークサイズの確認を実施して、（ｉ）PCR増幅産物が理論増幅産物長から±20%以上乖離がなく、シングルピークであること、および（ｉｉ）TapeStation濃度で20ng以上、濃度１ng/μL以上であること、を各々確認した。 Each of the PCR products was purified using AMPure XP (Beckman Coulter), and the concentration and peak size were confirmed using Agilent 2200/4200 TapeStation. It was confirmed that there was no deviation of % or more, that there was a single peak, and (ii) that the TapeStation concentration was 20 ng or more and the concentration was 1 ng/μL or more.
実施例４：サンガーシーケンス
　実施例３において調製された各精製PCR産物を用いて、サンガーシーケンスを行った。 Example 4: Sanger Sequencing Using each purified PCR product prepared in Example 3, Sanger sequencing was performed.
　96ウェルプレートの各ウェルに、精製PCR産物10～90ng/ウェルおよびプライマー（実施例３において使用された各プライマー）7.5pmol/ウェルを添加し、合計容量15μL/ウェルとなるようNuclease Free水を添加し、各々調製鋳型液とした。 Add 10-90 ng/well of purified PCR product and 7.5 pmol/well of primer (each primer used in Example 3) to each well of a 96-well plate, and add Nuclease Free water to a total volume of 15 μL/well. Each was used as a prepared mold solution.
　当該各調製鋳型液5μLに、BigDye Terminator v3.1 Cycle sequencing Kit 5μLを添加して混合し、サーマルサイクラー（タカラバイオ社）にセットし、表５に示される条件にて、シーケンス反応を実施した。 To 5 μL of each prepared template solution, 5 μL of BigDye Terminator v3.1 Cycle sequencing Kit was added and mixed, set in a thermal cycler (Takara Bio Inc.), and a sequencing reaction was performed under the conditions shown in Table 5.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　Sephadex G-50 Fine（Cytiva社）を膨潤させた精製用プレートを洗浄し、シーケンス反応後の溶液を精製用プレートに添加後、遠心分離して精製反応液を回収した。精製反応液に10μL/ウェルで大塚蒸留水を添加したものをシーケンスプレートとした。 A purification plate with swollen Sephadex G-50 Fine (Cytiva) was washed, and the solution after the sequence reaction was added to the purification plate, followed by centrifugation to collect the purified reaction solution. A sequencing plate was prepared by adding 10 μL/well of Otsuka distilled water to the purified reaction solution.
　当該シーケンスプレートを、DNAシーケンサー：Applied Biosystems 3730xl DNA Analyzer（Applied Biosystems社）にセットし、シーケンス解析（FastRun（約1時間、 ～650塩基；Sequencing Analysis Software v6.0）でベースコールを実施、MixedPeak threshold 25%（デフォルト値）でヘテロコールを実施）を実施した。 The sequence plate was set in a DNA sequencer: Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems), and base calling was performed using sequence analysis (FastRun (approximately 1 hour, ~650 bases; Sequencing Analysis Software v6.0), MixedPeak threshold A heterocall was performed at 25% (default value).
実施例５：STING1遺伝子のSNPsのジェノタイプ検出
　実施例４において取得したDNA配列情報の末端部分をトリミングした後、測定遺伝子領域の参照配列にアライメントし、相同性を確認した。相同性の基準を満たしたサンプル についてSNP検出を行った。 Example 5: Genotype detection of SNPs of STING1 gene After trimming the terminal portion of the DNA sequence information obtained in Example 4, it was aligned to the reference sequence of the measured gene region to confirm homology. SNP detection was performed on samples that met the homology criteria.
　実施例４で取得したab1ファイルを、Chromas 2.6.6ソフトウェアのTrim Low Quality機能（設定値：Trim until average quality of 10 bases exceeds 15（デフォルト値））を用いてトリミングした塩基配列情報およびクオリティスコアをfastq形式で出力した。なお、Reverseプライマーより得られたデータについては、ChromasソフトウェアのReverse complement機能を使用し、相補鎖配列としたうえでトリミングを行った。Biopython 1.79のSeqIOモジュールを用いたスクリプトにより、塩基配列情報の末端50bpをトリミングし、fastq形式で出力して、さらに、トリミングした結果からSeqKit 2.1.0によりQV値が30以上の塩基の割合を算出した。SeqKitを用いてfastq形式からfasta形式ファイルに変換後、BLAST+2.12.0を用いて、測定遣伝子領域の配列とアライメントし、STING1遺伝子配列（NCBI Gene ID: 340061）との相同性が98％以上かつアラインメント領域が測定配列の98%以上をカバーしていること、および塩基配列データにおいてQV値が30以上である塩基割合が95%以上であることを確認した。同品質基準を満たしたab1ファイルより、Chromasを用いて測定SNPs（rs7380824、rs1131769、rs78233829およびrs11554776）のジェノタイプを検出した。 The ab1 file obtained in Example 4 was trimmed using the Trim Low Quality function of Chromas 2.6.6 software (setting value: Trim until average quality of 10 bases exceeds 15 (default value)) and the base sequence information and quality score were trimmed. Output in fastq format. The data obtained from the Reverse primer was trimmed using the Reverse complement function of Chromas software to obtain a complementary strand sequence. A script using Biopython 1.79's SeqIO module trims the terminal 50 bp of base sequence information, outputs it in fastq format, and then calculates the percentage of bases with a QV value of 30 or higher using SeqKit 2.1.0 from the trimmed results. did. After converting from fastq format to fasta format file using SeqKit, the sequence of the measured gene region was aligned using BLAST+2.12.0, and the homology with the STING1 gene sequence (NCBI Gene ID: 340061) was found to be 98 It was confirmed that the alignment region covered 98% or more of the measured sequence, and that the proportion of bases with a QV value of 30 or more in the base sequence data was 95% or more. Genotypes of measured SNPs (rs7380824, rs1131769, rs78233829 and rs11554776) were detected using Chromas from ab1 files that met the same quality standards.
　ヒト血液64検体から抽出したDNA 64サンプルを用いてサンガーシーケンシングによるSTING1遺伝子の４つのSNPs（rs7380824、rs1131769、rs78233829およびrs11554776）の測定精度を検証したところ、各SNPを挟む領域のPCR産物は、実施例３において定められた基準を全サンプルで満たしていた。全1,152個のサンガーシーケンシング結果（64血液検体に対して３領域のPCR産物でForwardとReverseプライマーで両側からシーケンシングし、室内再現性試験を３回実施）のうち、1,150解析において本実施例で定めた品質基準を満たしており、Reverseプライマーからの解析不良の２解析についても、Forwardプライマーの解析で十分補完可能で、非常に高い精度を誇る検証結果が得られた。 When we verified the measurement accuracy of four SNPs (rs7380824, rs1131769, rs78233829 and rs11554776) of the STING1 gene by Sanger sequencing using 64 DNA samples extracted from 64 human blood samples, we found that the PCR products in the region sandwiching each SNP were: All samples met the criteria established in Example 3. Out of a total of 1,152 Sanger sequencing results (64 blood samples were sequenced from both sides with forward and reverse primers using PCR products from 3 regions, and an in-house reproducibility test was performed 3 times), 1,150 were analyzed using this example. It met the quality standards established by the Forward primer, and the two failure analyzes from the Reverse primer could be fully complemented by the Forward primer analysis, resulting in extremely high accuracy verification results.
実施例６：逆転写反応によるcDNA調製
　実施例２で調製されたTotal RNA溶液（Total RNA 100ng含有）に、dNTP Mixture（各10mM）（PrimeScript RT-PCR kit（タカラバイオ社）に含まれる試薬）１μLおよびOligo dT Primer（2.5μM）（PrimeScript RT-PCR kitに含まれる試薬）１μLを混合し、合計容量10μLとなるようRNase Free 水を添加した。当該混合液をVeriti 200サーマルサイクラーにセットし、アニーリング反応（65℃で5分間；４℃で保持）を行った。 Example 6: cDNA preparation by reverse transcription reaction dNTP Mixture (10mM each) (reagent included in PrimeScript RT-PCR kit (Takara Bio Inc.)) was added to the Total RNA solution (containing 100ng of total RNA) prepared in Example 2. 1 μL and 1 μL of Oligo dT Primer (2.5 μM) (a reagent included in the PrimeScript RT-PCR kit) were mixed, and RNase Free water was added to make a total volume of 10 μL. The mixed solution was set in a Veriti 200 thermal cycler, and an annealing reaction (at 65°C for 5 minutes; held at 4°C) was performed.
　当該反応液に、5×PrimeScript Buffer（PrimeScript RT-PCR kitに含まれる試薬）４μL、RNase Inhibitor（40 U/μL）(同左)0.5μL、PrimeScript RTase（for 2 step）（同左）0.5μLおよびRNase Free 水 ５μLの混合液10μLを添加し、混合したのち、Veriti 200サーマルサイクラーにセットし、RT反応（42℃で30分間、次に70℃で15分間、その後、４℃で保持する）を実施した。 Add 4 μL of 5× PrimeScript Buffer (reagent included in PrimeScript RT-PCR kit), 0.5 μL of RNase Inhibitor (40 U/μL) (same as left), 0.5 μL of PrimeScript RTase (for 2 step) (same as left) to the reaction solution, and RNase. Add 10 μL of a mixture of 5 μL of free water, mix, set in a Veriti 200 thermal cycler, and perform RT reaction (30 minutes at 42℃, then 15 minutes at 70℃, then held at 4℃) did.
実施例７：Miseq SystemによるシーケンスのためのPCR産物の調製
　複数のSNPを挟む領域のPCR産物を各々調製した。 Example 7: Preparation of PCR products for sequencing using the Miseq System PCR products of regions sandwiching multiple SNPs were each prepared.
　当該PCRに使用されたプライマーペアのうち、当該４個のSNPを挟むプライマーペアとして、各々配列番号１および２からなるプライマー、rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペアとして、各々配列番号７および８からなるプライマー、rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペアとして、各々配列番号１３および１４からなるプライマーが各々使用された（図２）。なお、以下、各々配列番号１および２からなるプライマーにより挟まれる領域を「領域５」とし、各々配列番号７および８からなるプライマーにより挟まれる領域を「領域６」とし、各々配列番号１３および１４からなるプライマーにより挟まれる領域を「領域７」とする（図４参照）。 Among the primer pairs used for the PCR, the primer pairs sandwiching the four SNPs were the primers consisting of SEQ ID NOs: 1 and 2, and the primer pairs sandwiching the SNPs of rs7380824, rs1131769, and rs78233829 were SEQ ID NOs: 7 and 2, respectively. Primers consisting of SEQ ID NOs: 13 and 14 were used as primer pairs sandwiching the SNPs of rs1131769, rs78233829, and rs11554776, respectively (FIG. 2). In addition, hereinafter, the region sandwiched by the primers consisting of SEQ ID NOs: 1 and 2, respectively, will be referred to as "region 5", the area sandwiched by the primers consisting of SEQ ID NOs: 7 and 8, respectively, will be referred to as "region 6", and the region sandwiched by the primers consisting of SEQ ID NOs: 13 and 14, respectively. The region sandwiched by the primers consisting of the following is referred to as "region 7" (see FIG. 4).
　実施例６で調製された反応液（cDNA溶液）５～20μLに、2×Gflex PCR Buffer（Mg2+, dNTP plus）25μL、前記各プライマーペア溶液（forward primerおよびReverse primer）各10pmol相当量およびTks Gflex DNA Polymerase（1.25 units/μL）１μLを混合し、合計容量50μLとなるよう、Nuclease Free水を添加し、混合して、各PCR反応液を調製した。 To 5 to 20 μL of the reaction solution (cDNA solution) prepared in Example 6, add 25 μL of 2× Gflex PCR Buffer (Mg 2+ , dNTP plus), 10 pmol equivalent of each of the above primer pair solutions (forward primer and reverse primer), and Each PCR reaction solution was prepared by mixing 1 μL of Tks Gflex DNA Polymerase (1.25 units/μL), adding Nuclease Free water to a total volume of 50 μL, and mixing.
　当該PCR反応液を、Veriti 200 サーマルサイクラーにセットし、上表４に示されるPCR条件にて、PCRを実施した。当該各PCR産物を各々AMPure XP（Beckman Coulter社）により精製し、Agilent2200/4200 TapeStationを用いて濃度およびピークサイズの確認を実施して、収量および品質を確認した。その結果、（ｉ）ターゲットPCR増幅産物がPrimer Dimerを除くピークが確認できること、および（ｉｉ）TapeStation濃度で40ng以上、濃度１ng/μL以上であること、を各々確認した。 The PCR reaction solution was set in a Veriti 200 thermal cycler, and PCR was performed under the PCR conditions shown in Table 4 above. Each of the PCR products was purified using AMPure XP (Beckman Coulter), and concentration and peak size were confirmed using Agilent 2200/4200 TapeStation to confirm yield and quality. As a result, it was confirmed that (i) a peak of the target PCR amplification product excluding Primer Dimer could be confirmed, and (ii) the TapeStation concentration was 40 ng or more and the concentration was 1 ng/μL or more.
　当該各PCR産物（領域５、領域６および領域７の各PCR産物）の濃度が同じになるようElution Buffer（Buffer EB, QIAGEN社）に希釈し、領域５、領域６および領域７の各PCR産物を２：１：１の割合で混合した。当該混合PCR産物（10ng）に、Template Preparation D Buffer（ThruPLEX DNA-Seq Kit（タカラバイオ社）に含まれる試薬）２μLとTemplate Preparation D Enzyme（同左）１μLとの混合液３μLを添加し、混合した。当該混合液をVeriti 200サーマルサイクラーにセットし、反応（22℃で25分間、次に55℃で20分間、その後、４℃で保持する）を開始した。 Dilute each PCR product (each PCR product of region 5, region 6, and region 7) in Elution Buffer (Buffer EB, QIAGEN) so that the concentration is the same, and add each PCR product of region 5, region 6, and region 7. were mixed in a ratio of 2:1:1. To the mixed PCR product (10 ng), 3 μL of a mixture of 2 μL of Template Preparation D Buffer (reagent included in ThruPLEX DNA-Seq Kit (Takara Bio Inc.)) and 1 μL of Template Preparation D Enzyme (same as on the left) was added and mixed. . The mixture was placed in a Veriti 200 thermal cycler and the reaction was started (25 minutes at 22°C, then 20 minutes at 55°C, then held at 4°C).
　当該反応液に、Library Synthesis D Buffer（ThruPLEX DNA-Seq Kitに含まれる試薬）1μLとLibrary Synthesis D Enzyme（同左）１μLとの混合液２μLを添加し、混合して、当該混合液をVeriti 200サーマルサイクラーにセットし、反応（22℃で40分間、その後、４℃で保持する）を開始した。 Add 2 μL of a mixture of 1 μL of Library Synthesis D Buffer (reagent included in ThruPLEX DNA-Seq Kit) and 1 μL of Library Synthesis D Enzyme (same as left) to the reaction solution, mix, and transfer the mixture to Veriti 200 thermal The cycler was set and the reaction (40 minutes at 22°C, then held at 4°C) was started.
　さらに、当該反応液に、Library Amplification D Buffer（ThruPLEX DNA-Seq Kitに含まれる試薬）25μL、Library Amplification Enzyme（同左）１μLおよびNuclease Free水４μLの混合液30μLならびに内部標準溶液５μLを添加して、混合し、当該混合液をVeriti 200サーマルサイクラーにセットし、表６に示される条件にて反応を開始した。 Furthermore, 30 μL of a mixture of 25 μL of Library Amplification D Buffer (reagent included in ThruPLEX DNA-Seq Kit), 1 μL of Library Amplification Enzyme (same as left), and 4 μL of Nuclease Free water, and 5 μL of internal standard solution were added to the reaction solution. The mixture was placed in a Veriti 200 thermal cycler, and the reaction was started under the conditions shown in Table 6.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　当該反応産物をAMPure XPにより精製し、D1000 Screen TapeおよびD1000試薬キットとAgilent2200/4200 TapeStationを用いて増幅ピークサイズおよび濃度を測定した。ライブラリーのメインピークは想定されたPCR増幅長のものが確認され、ライブラリー濃度は２nM以上であることが確認された。 The reaction product was purified using AMPure XP, and the amplified peak size and concentration were measured using D1000 Screen Tape, D1000 Reagent Kit, and Agilent 2200/4200 TapeStation. The main peak of the library was confirmed to have the expected PCR amplification length, and the library concentration was confirmed to be 2 nM or more.
実施例８：Miseq Systemによる高速シーケンス
　Miseq System（Illumina社）を用いて、実施例７で調製されたライブラリーのシーケンス解析を実施した。 Example 8: High-speed sequencing using Miseq System Sequence analysis of the library prepared in Example 7 was performed using Miseq System (Illumina).
　作製したライブラリーとPhiX Control v3を３：２の割合で混合し、Miseq System（Illumina社）を用いて300 base両末端シーケンス解析を実施した。シーケンサー付属のソフトウェア（MiSeq Control Software v4.0.0.1769, Real Time Analysis v1.18.54.4, bcl2fastq2v2.17）により塩基配列（リード配列）を取得した。 The prepared library and PhiX Control v3 were mixed at a ratio of 3:2, and 300 base both-end sequence analysis was performed using Miseq System (Illumina). The nucleotide sequence (read sequence) was obtained using the software included with the sequencer (MiSeq Control Software v4.0.0.1769, Real Time Analysis v1.18.54.4, bcl2fastq2v2.17).
実施例９：STING1遺伝子のSNPsのハプロタイプ検出
　Miseq Systemによって取得した塩基配列データを、STAR（version 2.7.9a）を用いて、ヒトゲノム参照配列 GRCh38（GENCODE）にマッピングし、FreeBayes（version 1.3.5）を用いてハプロタイプの変異検出解析を実施した。解析には測定SNPがQV40以上の塩基を使用し、ヘテロコールの閾値は、Allele frequency > 0.1とした。出力されたVCFファイルについてVcflib（version 1.0.2）を使用し、マルチアレルを単一のコールに分解して、VCFtools（version 0.1.16）を用いてコールされたポジションを測定SNPのみに絞り込み、また、マルチアレルでコールされた場合は、bcftools（version 1.12）で個々のアレルに分解した。WhatsHap（version 1.0）を使用して検出された測定SNPsのハプロタイプフェージングを行い、bcftools（version 1.12）を用いて個別のvcfファイルをマージして、ハプロタイプの結果を取得した。 Example 9: Detection of haplotypes of SNPs in the STING1 gene The nucleotide sequence data obtained by the Miseq System was mapped to the human genome reference sequence GRCh38 (GENCODE) using STAR (version 2.7.9a), and was mapped to the human genome reference sequence GRCh38 (GENCODE) using FreeBayes (version 1.3.5). Haplotype mutation detection analysis was performed using . Bases with measured SNPs of QV40 or higher were used for analysis, and the heterocall threshold was set to Allele frequency > 0.1. For the output VCF file, use Vcflib (version 1.0.2) to decompose multi-alleles into single calls, and use VCFtools (version 0.1.16) to narrow down the called positions to only the measured SNPs. In addition, when multiple alleles were called, they were broken down into individual alleles using bcftools (version 1.12). Haplotype phasing of the detected measured SNPs was performed using WhatsHap (version 1.0), and separate vcf files were merged using bcftools (version 1.12) to obtain haplotype results.
　実施例５および本実施例によって得られたジェノタイプおよびハプロタイプの結果より、最終的な測定SNPsのハプロタイプを決定した。 From the genotype and haplotype results obtained in Example 5 and this example, the final haplotype of the measured SNPs was determined.
　なお、実施例５および本実施例に従い行われた、ヒト血液64検体から抽出したDNAおよびRNA各64サンプルを用いて作業日を変えた全３回の試行によって得られた結果より、同一サンプル由来のハプロタイプはすべての試行で同一のハプロタイプが得られており、本測定系の非常に高い室内再現精度が示された。 In addition, from the results obtained in a total of three trials conducted in accordance with Example 5 and this example using 64 samples each of DNA and RNA extracted from 64 human blood samples and on different working days, it was found that the samples were derived from the same sample. The same haplotype was obtained in all trials, demonstrating the extremely high intra-laboratory reproducibility of this measurement system.
　本発明のヒトSTING1遺伝子に関するハプロタイプ解析法は、STING作動薬による治療が求められる患者選択に有用である。 The haplotype analysis method for the human STING1 gene of the present invention is useful for selecting patients who require treatment with STING agonists.

Claims (20)

  1. rs7380824、rs1131769、rs78233829およびrs11554776によって各々特定される、ヒトSTING1遺伝子における４個の一遺伝子多型(SNP)の組み合わせに基づくハプロタイプを決定するための方法であって、
    （ａ）ヒトの生体試料からゲノムDNAおよびRNAを各々抽出する工程、
    （ｂ）前記（ａ）記載の工程において抽出されたRNAから、RT反応により、cDNAを調製する工程、
    （ｃ）（ｉ）前記（ｂ）記載の工程において調製されたcDNA、および（ｉｉ）当該４個のSNPのうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
    （ｄ）前記（ｃ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、
    （ｅ）（ｉ）前記（ａ）記載の工程において抽出されたゲノムDNA、および（ｉｉ）当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
    （ｆ）前記（ｅ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、ならびに
    （ｇ）（ｉ）前記（ｄ）記載の工程において取得されたDNA配列情報から、ヒトSTING1遺伝子の一以上の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出し、（ｉｉ）前記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出し、（ｉｉｉ）前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程において抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の対立遺伝子のハプロタイプを決定する工程を含む、当該方法。     A method for determining a haplotype based on a combination of four monogenic polymorphisms (SNPs) in the human STING1 gene, each identified by rs7380824, rs1131769, rs78233829 and rs11554776, comprising:
    (a) Extracting each of genomic DNA and RNA from a human biological sample;
    (b) a step of preparing cDNA by RT reaction from the RNA extracted in the step described in (a) above;
    (c) (i) cDNA prepared in the step described in (b) above, and (ii) a set designed to sandwich at least three consecutive SNPs among the four SNPs. Using the above primer pairs, performing PCR separately for each primer pair,
    (d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof;
    (e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, The process of performing PCR separately,
    (f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above. From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. The method comprises determining the haplotypes of both alleles of the human STING1 gene by complementing the information.
  2. ヒトの生体試料が全血である、請求項１記載の方法。 2. The method of claim 1, wherein the human biological sample is whole blood.
  3. 請求項１（ｃ）（ｉｉ）記載の一組以上のプライマーペアが、（ａ）当該４個のSNPすべてを挟むプライマーペア、（ｂ）rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペア、ならびに（ｃ）rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペアである、請求項１または２記載の方法。 The one or more primer pairs according to claim 1(c)(ii) include (a) a primer pair sandwiching all four SNPs, (b) a primer pair sandwiching SNPs rs7380824, rs1131769, and rs78233829, and ( c) The method according to claim 1 or 2, which is a primer pair sandwiching SNPs of rs1131769, rs78233829, and rs11554776.
  4. 当該４個のSNPすべてを挟むプライマーペアが、
    （ｉ）配列番号１および２、（ｉｉ）配列番号３および４、ならびに（ｉｉｉ）配列番号５および６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項３記載の方法。     The primer pair sandwiching all four SNPs is
    Claim 3, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 1 and 2, (ii) SEQ ID NO: 3 and 4, and (iii) SEQ ID NO: 5 and 6. Method described.
  5. rs7380824、rs1131769およびrs78233829のSNPを挟むプライマーペアが、
    （ｉ）配列番号７および８、（ｉｉ）配列番号９および１０、ならびに（ｉｉｉ）配列番号１１および１２から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項３記載の方法。     The primer pair sandwiching the SNPs of rs7380824, rs1131769 and rs78233829 was
    Claim 3, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 7 and 8, (ii) SEQ ID NO: 9 and 10, and (iii) SEQ ID NO: 11 and 12. Method described.
  6. rs1131769、rs78233829およびrs11554776のSNPを挟むプライマーペアが、
    （ｉ）配列番号１３および１４、（ｉｉ）配列番号１５および１６、ならびに（ｉｉｉ）配列番号１７および１８から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項３記載の方法。     The primer pair sandwiching the SNPs of rs1131769, rs78233829 and rs11554776 was
    Claim 3, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 13 and 14, (ii) SEQ ID NO: 15 and 16, and (iii) SEQ ID NO: 17 and 18. Method described.
  7. 請求項１（ｂ）記載のRT反応が、抽出されたRNAを約65℃で約５分間、その後、約４℃で保持したのち、約42℃で約30分間、続いて、約70℃で約15分間、その後、約４℃で保持することで実施される、請求項１～６の何れか一項記載の方法。 The RT reaction according to claim 1(b) comprises heating the extracted RNA at about 65°C for about 5 minutes, then holding at about 4°C, then at about 42°C for about 30 minutes, and then at about 70°C. 7. A method according to any one of claims 1 to 6, carried out for about 15 minutes, followed by holding at about 4°C.
  8. 請求項１（ｃ）記載のPCRが、表１に示される条件で実施される、請求項１～７の何れか一項記載の方法。
    Figure JPOXMLDOC01-appb-T000001
         The method according to any one of claims 1 to 7, wherein the PCR according to claim 1(c) is performed under the conditions shown in Table 1.
    Figure JPOXMLDOC01-appb-T000001
  9. 請求項１（ｄ）記載のシーケンシングが、次世代シーケンシングで実施される、請求項１～８の何れか一項記載の方法。 The method according to any one of claims 1 to 8, wherein the sequencing according to claim 1(d) is performed by next generation sequencing.
  10. 次世代シーケンシングが、パイロシーケンシング、合成によるシーケンシング、Ion Torrent（登録商標）半導体シーケンシングまたはライゲーションによるシーケンシングである、請求項９記載の方法。 10. The method of claim 9, wherein the next generation sequencing is pyrosequencing, sequencing by synthesis, Ion Torrent® semiconductor sequencing or sequencing by ligation.
  11. 次世代シーケンシングが合成によるシーケンシングであり、当該合成によるシーケンシングが、MiSeq（登録商標）（Illumina社）を用いた手法で実施される、請求項９記載の方法。 10. The method according to claim 9, wherein the next generation sequencing is synthetic sequencing, and the synthetic sequencing is performed using MiSeq (registered trademark) (Illumina).
  12. 請求項１（ｅ）（ｉｉ）記載の４個のSNPのうちの一以上のSNPを各々挟むプライマーペアが、（ａ）rs7380824のSNPを挟むプライマーペア、（ｂ）rs1131769およびrs78233829の二つのSNPを挟むプライマーペアならびに（ｃ）rs11554776のSNPを挟むプライマーペアからなる、請求項１～１１の何れか一項記載の方法。 The primer pair each sandwiching one or more SNPs among the four SNPs described in claim 1(e)(ii) is (a) a primer pair sandwiching an SNP of rs7380824, (b) two SNPs of rs1131769 and rs78233829. and (c) a primer pair sandwiching the SNP of rs11554776.
  13. rs7380824のSNPを挟むプライマーペアが、
    （ｉ）配列番号１９および２０、（ｉｉ）配列番号２１および２２、ならびに（ｉｉｉ）配列番号２３および２４から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項１２記載の方法。     The primer pair that spans the SNP of rs7380824 is
    Claim 12, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 19 and 20, (ii) SEQ ID NO: 21 and 22, and (iii) SEQ ID NO: 23 and 24. Method described.
  14. rs1131769およびrs78233829の二つのSNPを挟むプライマーペアが、
    （ｉ）配列番号２５および２６、（ｉｉ）配列番号２７および２８、ならびに（ｉｉｉ）配列番号２９および３０から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項１２記載の方法。     A primer pair sandwiching two SNPs, rs1131769 and rs78233829,
    Claim 12, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 25 and 26, (ii) SEQ ID NO: 27 and 28, and (iii) SEQ ID NO: 29 and 30. Method described.
  15. rs11554776のSNPを挟むプライマーペアが、
    （ｉ）配列番号３１および３２、（ｉｉ）配列番号３３および３４、ならびに（ｉｉｉ）配列番号３５および３６から選択される一組の塩基配列からなる一本鎖DNAの組み合わせである、請求項１２記載の方法。     The primer pair that sandwiches the SNP of rs11554776 is
    Claim 12, which is a combination of single-stranded DNA consisting of a set of base sequences selected from (i) SEQ ID NO: 31 and 32, (ii) SEQ ID NO: 33 and 34, and (iii) SEQ ID NO: 35 and 36. Method described.
  16. 請求項１（ｅ）記載のPCRが、請求項８記載の表１に示される条件で実施される、請求項１～１５の何れか一項記載の方法。 The method according to any one of claims 1 to 15, wherein the PCR according to claim 1(e) is performed under the conditions shown in Table 1 according to claim 8.
  17. 請求項１（ｆ）記載のシーケンシングが、サンガーシーケンシングである、請求項１～１６の何れか一項記載の方法。 The method according to any one of claims 1 to 16, wherein the sequencing according to claim 1(f) is Sanger sequencing.
  18. rs7380824、rs1131769、rs78233829およびrs11554776によって各々特定される、ヒトSTING1遺伝子における４個の一遺伝子多型(SNP)の組み合わせに基づくハプロタイプを決定するための方法であって、
    （ａ）ヒトの生体試料からゲノムDNAおよびRNAを各々抽出する工程、
    （ｂ）前記（ａ）記載の工程において抽出されたRNAから、RT反応により、cDNAを調製する工程、
    （ｃ）（ｉ）前記（ｂ）記載の工程において調製されたcDNA、および（ｉｉ）当該４個のSNPのうち、隣接して連続する少なくとも３個のSNPを挟むように設計された一組以上のプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
    （ｄ）前記（ｃ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、
    （ｅ）（ｉ）前記（ａ）記載の工程において抽出されたゲノムDNA、および（ｉｉ）当該４個のSNPのうちの一以上のSNPを各々挟むプライマーペアを用いて、当該プライマーペアごとに別々にPCRを行う工程、
    （ｆ）前記（ｅ）記載の工程において増幅された各PCRアンプリコンについて、シーケンシングを各々行い、それらのDNA配列情報を取得する工程、ならびに
    （ｇ）（ｉ）前記（ｄ）記載の工程において取得されたDNA配列情報から、ヒトSTING1遺伝子の一以上の対立遺伝子について、当該４個のSNPの組み合わせに基づくハプロタイプを抽出し、（ｉｉ）前記（ｆ）記載の工程において取得されたDNA配列情報から、当該４個の各SNPにおけるジェノタイプを各々抽出し、（ｉｉｉ）前記（ｉ）記載の工程において抽出されたハプロタイプに関する情報を、前記（ｉｉ）記載の工程において抽出されたジェノタイプに関する情報によって補完することで、ヒトSTING1遺伝子の両方の対立遺伝子のハプロタイプを決定する工程を含み、
    ここで、前記（ｂ）記載のRT反応が、抽出されたRNAを約65℃で約５分間、その後、約４℃で保持したのち、約42℃で約30分間、続いて、約70℃で約15分間、その後、約４℃で保持することで実施され、
    前記（ｃ）（ｉｉ）記載の一組以上のプライマーペアが、（ａ）配列番号１および２の一組の塩基配列からなる一本鎖DNAの組み合わせ、（ｂ）配列番号７および８の一組の塩基配列からなる一本鎖DNAの組み合わせ、ならびに（ｃ）配列番号１３および１４の一組の塩基配列からなる一本鎖DNAの組み合わせであり、
    前記（ｃ）記載のPCRが、表２に示される条件で実施され、
    Figure JPOXMLDOC01-appb-T000002
     前記（ｄ）記載のシーケンシングが、MiSeq（登録商標）（Illumina社）を用いた手法で実施され、
    前記（ｅ）（ｉｉ）記載の４個のSNPのうちの一以上のSNPを各々挟むプライマーペアが、（ａ）配列番号２１および２２の一組の塩基配列からなる一本鎖DNAの組み合わせ、（ｂ）配列番号２７および２８の一組の塩基配列からなる一本鎖DNAの組み合わせ、ならびに（ｃ）配列番号３３および３４の一組の塩基配列からなる一本鎖DNAの組み合わせからなり、
    前記（ｅ）記載のPCRが、上表２に示される条件で実施され、
    前記（ｆ）記載のシーケンシングが、サンガーシーケンシングである、当該方法。     A method for determining a haplotype based on a combination of four monogenic polymorphisms (SNPs) in the human STING1 gene, each identified by rs7380824, rs1131769, rs78233829 and rs11554776, comprising:
    (a) Extracting each of genomic DNA and RNA from a human biological sample;
    (b) a step of preparing cDNA by RT reaction from the RNA extracted in the step described in (a) above;
    (c) (i) cDNA prepared in the step described in (b) above, and (ii) a set designed to sandwich at least three consecutive SNPs among the four SNPs. Using the above primer pairs, performing PCR separately for each primer pair,
    (d) Sequencing each PCR amplicon amplified in the step described in (c) above, and obtaining DNA sequence information thereof;
    (e) Using (i) the genomic DNA extracted in the step described in (a) above, and (ii) primer pairs that sandwich one or more of the four SNPs, for each primer pair, The process of performing PCR separately,
    (f) Sequencing each PCR amplicon amplified in the step described in (e) above and obtaining DNA sequence information thereof, and (g) (i) Step described in (d) above. From the DNA sequence information obtained in step (f), extract a haplotype based on the combination of the four SNPs for one or more alleles of the human STING1 gene, and (ii) extract the DNA sequence obtained in the step described in (f) above. From the information, extract the genotypes for each of the four SNPs, and (iii) extract the information regarding the haplotypes extracted in the step described in (i) above, and the information regarding the genotypes extracted in the step described in (ii) above. determining the haplotypes of both alleles of the human STING1 gene, supplemented with information;
    Here, in the RT reaction described in (b) above, the extracted RNA is heated at about 65°C for about 5 minutes, then held at about 4°C, then at about 42°C for about 30 minutes, and then at about 70°C. for about 15 minutes, then held at about 4°C,
    The one or more primer pairs described in (c)(ii) above are (a) a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 1 and 2, (b) one of SEQ ID NOs: 7 and 8; A combination of single-stranded DNA consisting of a set of base sequences, and (c) a combination of single-stranded DNA consisting of a set of base sequences of SEQ ID NOs: 13 and 14,
    The PCR described in (c) above is performed under the conditions shown in Table 2,
    Figure JPOXMLDOC01-appb-T000002
     The sequencing described in (d) above is performed using a method using MiSeq (registered trademark) (Illumina),
    A pair of primers each sandwiching one or more SNPs among the four SNPs described in (e) and (ii) above is (a) a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 21 and 22; (b) a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 27 and 28, and (c) a combination of single-stranded DNA consisting of a pair of base sequences of SEQ ID NOs: 33 and 34,
    The PCR described in (e) above is carried out under the conditions shown in Table 2 above,
    The method described in (f) above, wherein the sequencing described in (f) is Sanger sequencing.
  19. 配列番号１～１８からなる群から選択される塩基配列からなる一本鎖DNA。 Single-stranded DNA consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 18.
  20. 配列番号１９～３６からなる群から選択される塩基配列からなる一本鎖DNA。 Single-stranded DNA consisting of a base sequence selected from the group consisting of SEQ ID NOs: 19-36.
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JP2020517723A (en) * 2017-04-28 2020-06-18 アドゥロ バイオテック，インク． Bis 2'-5'-RR-(3'F-A)(3'F-A) cyclic dinucleotide compound and use thereof
WO2021250530A1 (en) * 2020-06-08 2021-12-16 Curadev Pharma Pvt. Ltd., Rapid method for genotyping sting variants in human individuals

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JP2020517723A (en) * 2017-04-28 2020-06-18 アドゥロ バイオテック，インク． Bis 2'-5'-RR-(3'F-A)(3'F-A) cyclic dinucleotide compound and use thereof
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