WO2018074458A1 - Procédé et agent de diagnostic - Google Patents

Procédé et agent de diagnostic Download PDF

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WO2018074458A1
WO2018074458A1 PCT/JP2017/037496 JP2017037496W WO2018074458A1 WO 2018074458 A1 WO2018074458 A1 WO 2018074458A1 JP 2017037496 W JP2017037496 W JP 2017037496W WO 2018074458 A1 WO2018074458 A1 WO 2018074458A1
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hepatitis
hla
virus
progression
dpb1
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PCT/JP2017/037496
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English (en)
Japanese (ja)
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徳永　勝士
裕美 澤井
雅史 溝上
奈央 西田
真也 杉山
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国立大学法人東京大学
国立研究開発法人国立国際医療研究センター
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Priority to JP2018546350A priority Critical patent/JPWO2018074458A1/ja
Publication of WO2018074458A1 publication Critical patent/WO2018074458A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a detection method and a diagnostic agent for HLA class II gene polymorphism involved in chronicity of hepatitis B and HLA gene polymorphism involved in hepatitis B-derived liver cancer.
  • Hepatitis B virus HBV
  • Hepatitis B virus HBV
  • Acute hepatitis develops symptoms after a latent period of 1 to 6 months after infection, and begins to recover within a few weeks.
  • 1-2% of patients who develop acute hepatitis are at risk of developing fulminant hepatitis, and 70-80% of those who develop fulminant hepatitis die.
  • infected HBV is not eliminated from the body, but becomes a carrier when it settles in the liver for more than 6 months.
  • 80 to 90% of carriers go through an asymptomatic period, a transient hepatitis stage, and a hepatitis sedation stage, and then continue as an asymptomatic carrier.
  • Chronic carriers of hepatitis B virus are distributed in Southeast Asia and the East Pacific region, and in particular, it is said that there are approximately 1.5 million hepatitis B infected people in Japan.
  • HLA-DPB1 and HLA-DQB1 are related to HBV persistent infection and virus elimination (Kamatani et al. 2009, Nishida et al. 2012). Further, although resistance and sensitive alleles have been investigated for HLA-DPB1, detailed studies on alleles and haplotypes have not been conducted on HLA-DRB1 and HLA-DQB1. In addition, GWAS has also been made for hepatitis B-derived liver cancer, and multiple genetic factors have been identified, but there are some that cannot be reproduced by the Japanese. There is a need.
  • the present invention performs analysis using the healthy subject group, chronic hepatitis B group and hepatitis B disease state progression group (liver cancer), and is sensitive and resistant to the progression of hepatitis B virus-related diseases.
  • An object of the present invention is to provide a method and a diagnostic agent for testing the ease of progression of a disease state when suffering from a hepatitis B virus-related disease by identifying alleles.
  • the following methods (1) to (12) are methods and diagnostic agents for testing the ease of progression of a disease state when suffering from a hepatitis B virus-related disease according to the present invention.
  • the subject has a step of comparing the subject's HLA type with a first criterion of ease of pathological progression when he / she suffers from a hepatitis B virus-related disease; To test the ease of progression of the pathological condition when suffering from the disease.
  • the method further includes a step of comparing the frequency of mutation of the hepatitis B virus population infected with the subject with a second standard of ease of progression of a disease state when suffering from a hepatitis B virus related disease. The method of (1) above.
  • the HLA type includes HLA-DPB1 * 02: 01
  • the method according to (1) above which is a criterion that it is difficult to progress to liver cancer when hepatitis B virus is persistently infected.
  • the HLA type includes HLA-DPB1 * 02: 01
  • the method according to (2) above which is a criterion that it is difficult to progress to liver cancer when hepatitis B virus is persistently infected.
  • the mutation is a mutation in the HBs antigen protein derived from the hepatitis B virus population and the second criterion is that the 166th most frequently occurring amino acid of the HBs antigen protein is not leucine
  • the standard is that it is difficult to develop into liver cancer when hepatitis virus is persistently infected, or if the 236th most frequently occurring amino acid of the HBs antigen protein is not glutamine, If it is a criterion that it is difficult to progress to liver cancer, or if the 251st most frequently occurring amino acid of the HBs antigen protein is not arginine, it is difficult to progress to liver cancer when hepatitis B virus is persistently infected.
  • hepatitis B A reference that when persistent infection in Luz hardly progress in liver cancer, the method according to (4).
  • the method according to (4) In the case where the pathological progress is progress to liver cancer when hepatitis B virus is persistently infected, and the first criterion is that the HLA type includes HLA-A * 33: 03
  • the pathological progression is progression to chronic hepatitis B in the case of persistent infection with hepatitis B virus
  • the first criterion is that the HLA type is HLA-DPB1 * 09: 01, HLA-DRB1 * In the case of including 15:02 and HLA-DQB1 * 06: 01, it is a criterion that it is easy to progress to chronic hepatitis B when it is persistently infected with hepatitis B virus, (1) or (2) The method described in 1.
  • the pathological progression is progression to chronic hepatitis B in the case of persistent infection with hepatitis B virus
  • the first criterion is that the HLA type is HLA-DPB1 * 04: 01, HLA-DRB1 * When 13:02 and HLA-DQB1 * 06: 04 are included, it is a criterion that it is difficult to develop chronic hepatitis B when persistent infection with hepatitis B virus occurs (1) or (2) The method described in 1.
  • (11) Primer set for detecting HLA type of HLA-DPB1 * 09: 01, HLA-DRB1 * 15: 02 and HLA-DQB1 * 06: 01, or HLA-DPB1 * 04: 01, HLA-DRB1 * 13: A diagnostic agent for testing the easiness of progression to chronic hepatitis B when persistently infected with hepatitis B virus, comprising a primer set for detecting HLA type 02 and HLA-DQB1 * 06: 04.
  • (12) The diagnostic agent according to (11), further comprising a primer set for determining the frequency of mutation in the hepatitis B virus population, or a specific binding substance for determining the frequency of mutation in the hepatitis B virus population.
  • the subject suffers from hepatitis B virus-related disease by comparing the subject's HLA type with the first criteria for ease of pathological progression when he / she suffers from hepatitis B virus-related disease.
  • a method and a diagnostic agent for testing the ease of progression of the disease state will be described.
  • the hepatitis B virus-related disease is at least one of persistent infection, asymptomatic carrier, chronic hepatitis B, cirrhosis and liver cancer.
  • the pathological progress when suffering from a hepatitis B virus-related disease is, for example, when a patient with persistent hepatitis B virus infection develops chronic hepatitis, when a chronic hepatitis patient develops cirrhosis, chronic hepatitis patient May develop liver cancer, patients with cirrhosis may develop liver cancer, or may develop liver cancer from asymptomatic carriers. Unlike patients suffering from hepatitis C virus, patients who are persistently infected with hepatitis B virus may progress to liver cancer without going through the pathological stage. Ease of pathological progress is whether or not pathological progress is easier or difficult compared with patients suffering from hepatitis B virus-related diseases.
  • a patient whose disease state is likely to progress is a patient who is likely to develop chronic hepatitis, cirrhosis or liver cancer when he / she is persistently infected with hepatitis B virus.
  • a patient who is difficult to progress is a patient who is less likely to develop chronic hepatitis, cirrhosis or liver cancer when he / she is persistently infected with hepatitis B virus.
  • the onset of the disease is as follows: mother-to-infant infection (vertical infection) from a person with persistent HBV infection; If blood or body fluid of a person with persistent HBV infection enters the body due to a medical action during the period; the result of infection with HBV while using an immunosuppressant or anticancer drug that reduces the body's immunity And HBV cannot be eliminated from the body, causing persistent infections; and healthy individuals have recently been infected with foreign species such as genotype A type Western and Asian / African type HBV. Thus, when infected with HBV, 80 to 90% become asymptomatic carriers, but 10 to 20% shift to chronic hepatitis. In addition, some of them shift to cirrhosis and liver cancer.
  • Liver cirrhosis refers to a condition in which fibers formed when the liver damaged by hepatitis B virus infection is repaired spread to the liver. As the liver becomes hard, ascites, esophageal varices occur, The decrease may cause hepatic encephalopathy or jaundice.
  • Hepatoma refers to hepatocellular carcinoma caused by hepatitis B virus infection.
  • hepatitis B virus-related diseases are susceptible to canceration due to progression of hepatitis B virus-related diseases (hepatitis B virus-related diseases are It refers to allyl that is pathologically advanced and susceptible to cancer) or resistant (hepatitis B virus-related disease is difficult to progress and cancerous).
  • genomic DNA was prepared from biological samples collected from chronic hepatitis B patients, liver cancer patients, or healthy individuals. It can be carried out by analyzing the gene sequence by a direct sequence method or the like. Since the novel allyl thus obtained was found in patients with chronic hepatitis B or liver cancer, it is related to canceration due to progression of hepatitis B virus-related disease. Is a promising candidate as an allyl according to this embodiment. In order to confirm the relationship between the allele selected as described above and canceration due to the progression of the disease state of the hepatitis B virus-related disease, statistical tests can be performed.
  • the appearance rate of the candidate allele in the group having chronicity of hepatitis B and the group of healthy persons is calculated, respectively, and the association between the candidate allele and chronicity of hepatitis B is statistically tested.
  • the test can be performed by a statistically appropriate method such as ⁇ 2 test, Fisher's exact test, etc., and the significance level may be corrected as necessary.
  • the method for detecting allyl is not particularly limited, and can be selected from methods known to those skilled in the art.
  • TaqMan PCR method MALDI-TOF / MS method, ASO (allele-specific oligonucleotide) method, direct sequencing method, RFLP method, invader method, TGGE, DGGE method, MutY enzyme method, microarray, Protein truncation test method , Snipper method, Luminex method, direct sequencing method, HLA Impression method, micro SSP method and the like can be selected according to the purpose.
  • the direct sequencing method may be a conventional direct sequencing method or a next generation direct sequencing method.
  • the HLA Imputation method is a method of genetic statistical analysis in which an individual's HLA genotype is estimated with high accuracy by a computer.
  • the micro SSP method is a method in which PCR amplification is performed using a plurality of primers, whether or not a characteristic polymorphic portion is amplified by gel electrophoresis, and alleles are determined based on the amplification pattern. is there.
  • the present inventors analyzed GWAS and HLA six loci by HLA imputation for 473 specimens of Japanese patients with hepatitis B positive liver cancer and 516 specimens of chronic hepatitis B patients and asymptomatic carrier group 516 specimens.
  • HLA-A * 33: 03 was identified as an allele susceptible to canceration due to the progression of hepatitis B virus-related disease.
  • the present inventors also confirmed that the HLA-DPB1 GWAS and HLA six loci for the HLA-DPB1 GHAS and HLA 6 loci of the Japanese hepatitis B positive liver cancer patient group 473 samples, the chronic hepatitis B patient group and the asymptomatic carrier group 516 samples.
  • HLA-DPB1 * 02: 01 was identified as an allele resistant to carcinogenesis due to the progression of hepatitis B virus-related disease.
  • HLA is a human major histocompatibility complex (MHC), a membrane protein that binds to foreign antigen peptides such as grafts, bacteria, and viruses and presents them to T cells. It is known that many alleles exist in HLA, and such information is described in HLA nomenclature (http://hla.alleles.org/announcement.html) and the like. In addition, the notation of allyl and polymorphism in this specification is based on the notation method by HLA nomenclature.
  • GenBank NIH genetic sequence database
  • DDBJ DNA Data Bank of Japan
  • IPD-IMGT / HLA Immno Polymorphism Database / p. / Wac. / Wac. / Data registered in a database such as imgt / hla /
  • GenBank the base sequence of HLA-A * 33: 03 cDNA is registered as, for example, HSU09740
  • the amino acid sequence thereof is registered as, for example, AAA79865.
  • GenBank registers the base sequence of HLA-DPB1 * 02: 01 cDNA, for example, AY804134, and the amino acid sequence, for example, AAW78739.
  • allyl HLA-A * 33: 03 and HLA-DPB1 * 02: 01 are referred to as “alleles related to canceration due to progression of hepatitis B virus-related disease”, “according to this embodiment "Allyl", or HLA type, polymorphism, or SNP instead of allyl.
  • the method according to the present embodiment is related to canceration due to progression of the pathological condition of hepatitis B virus-related disease.
  • the present invention relates to a method for detecting a predisposition to canceration caused by progression of a hepatitis B virus-related disease using allele (in the following description, sometimes referred to as “HLA type”, “polymorphism”, or “SNP”). .
  • HLA type hepatitis B virus-related disease using allele
  • SNP polymorphism
  • the following steps are included: a) comparing a subject's HLA type with a first criterion of ease of pathological progression when suffering from a hepatitis B virus-related disease.
  • the allele sensitive to canceration caused by the progression of the hepatitis B virus-related disease is HLA-A * 33: 03
  • the hepatitis B virus-related disease is the disease progression.
  • the allyl that is resistant to canceration is HLA-DPB1 * 02: 01. That is, the above first criterion is that when a subject has HLA-A * 33: 03 allele, hepatitis easily develops from hepatitis B virus-related disease and becomes cancerous.
  • DPB1 * 02: 01 allyl is present, it is a criterion that it is difficult to progress from hepatitis B virus-related disease to liver cancer. The above method will be described in detail below.
  • the step of comparing the subject's HLA type with the first criteria for the ease of progression of the disease state when he / she suffers from a hepatitis B virus-related disease is, in other words, HLA-A * 33: 03 and / or Alternatively, detection of alleles of the base sequence and amino acid sequence of HLA-DPB1 * 02: 01 allele.
  • Allele detection can be performed at the gene level or protein level.
  • genomic DNA or mRNA can be prepared from a specimen, and based on the nucleotide sequence, alleles related to canceration due to the progression of hepatitis B virus-related disease in the genomic DNA or mRNA can be detected.
  • Genomic DNA or mRNA prepared by a method well known to those skilled in the art can be used based on a biological sample collected from the specimen to be examined.
  • the biological sample collected from the specimen used in the method of the present embodiment was, for example, a cell or tissue of a blood cell specimen, hair, stool, urine, saliva, cells, cells scraped from the nasal mucosa, or scraped from the oral mucosa. Although a cell etc. can be used, it is not limited to these.
  • Genomic DNA can be prepared by any known method, and examples thereof include a phenol / chloroform method and a cetyltrimethylammonium bromide (CTAB) method.
  • mRNA can be prepared by any known method, such as the guanidine isothiocyanate method.
  • a commercially available kit may be used for the preparation of genomic DNA or mRNA. Examples of the kit include Wizard Genomic DNA Purification Kit (Promega) for preparing genomic DNA, and NucleoTrap mRNA Kit (Clontech) for preparing mRNA.
  • cDNA may be synthesized from mRNA for the detection of allele described below.
  • a cDNA synthesis method any method known in the art may be used.
  • cDNA can be synthesized from RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using random primers or poly-T primers.
  • the method for detecting HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele in the genomic DNA or mRNA prepared as described above is not particularly limited.
  • direct sequencing method polymerase chain reaction (PCR), restriction enzyme fragment length polymorphism (RFLP), hybridization method, primer extension reaction, mass spectroscopy, Luminex method, direct sequencing method, HLA Impression method, micro SSP method
  • PCR polymerase chain reaction
  • RFLP restriction enzyme fragment length polymorphism
  • hybridization method primer extension reaction
  • mass spectroscopy Luminex method
  • Luminex method direct sequencing method
  • HLA Impression method HLA Impression method
  • micro SSP method it is not limited to these methods.
  • HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele is detected by direct sequencing method using genomic DNA or cDNA derived from mRNA. Can do.
  • cDNA is prepared from the genomic DNA or mRNA prepared above; the region containing HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele to be detected is cloned into a vector. Or amplification by PCR; by determining the nucleotide sequence of the region. For example, it can be cloned by amplification by a PCR reaction using appropriate primers and ligation to an appropriate vector.
  • the vector can be subcloned into another vector, but is not limited thereto.
  • the vector include pBlue-Script TM SK (+) (Stratagene), pGEM-T (Promega), pAmp (TM: Gibco-BRL), p-Direct (Clontech), pCR2.1-TOPO (Invitrogene) ) And other commercially available plasmid vectors, viral vectors, artificial chromosome vectors and cosmid vectors.
  • the determination of the base sequence is not particularly limited, and examples thereof include, but are not limited to, a manual sequencing method using a radioactive marker nucleotide, an automatic sequencing method using a dye terminator, and a next-generation sequencing method. Based on the base sequence thus obtained, it is determined whether or not the specimen has a sequence corresponding to HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele.
  • the allele according to the present embodiment can also be detected using the PCR method.
  • PCR is performed using oligonucleotide primers that hybridize only to sequences having alleles according to this embodiment or sequences having other alleles.
  • the genomic DNA or mRNA derived from the specimen is amplified.
  • the specimen has the allyl according to the present embodiment homozygous, and PCR from the allele primer according to the present embodiment and other allele primers
  • the product is generated, the specimen has the allyl according to the present embodiment in a hetero form.
  • the other allyl primer generates a PCR product, it is indicated that the sample does not have the allyl according to the present embodiment.
  • the allele according to the present embodiment can also be detected using a restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • a region containing allyl according to the present embodiment to be detected is amplified by PCR.
  • the PCR product is cleaved with a restriction enzyme suitable for the allyl according to the present embodiment.
  • PCR products digested with restriction enzymes are separated by gel electrophoresis and visualized by ethidium bromide staining.
  • the allyl according to the present embodiment can also be detected using hybridization.
  • the hybridization method is a method for determining the presence or absence of an allyl according to this embodiment based on the property that a genomic DNA or mRNA derived from a specimen hybridizes with a complementary DNA molecule (for example, an oligonucleotide probe).
  • This hybridization method can be performed using various techniques for hybridization and detection such as known hybridization such as colony hybridization, plaque hybridization, Southern blotting and the like. The detailed procedure of the hybridization method is described in “Molecular Cloning, A Laboratory Manual 3rd ed.
  • hybridization can also be detected using a DNA chip.
  • an allyl-specific oligonucleotide probe according to the present embodiment is designed and affixed to a solid support. Then, the DNA sample derived from the specimen is brought into contact with the DNA chip to detect hybridization.
  • the TaqMan PCR method is a method in which an allele-specific Taqman probe and Taq polymerase are used to detect SNP and amplify a region containing SNP in parallel.
  • the Taqman probe is an oligonucleotide of about 20 bases labeled with a fluorescent substance at the 5 ′ end and a quencher at the 3 ′ end, and is designed to hybridize to a target SNP site.
  • Taq polymerase has 5′-3 ′ nuclease activity.
  • the Taqman probe When the allele region is amplified using a PCR primer designed to amplify the region containing the target allele in the presence of these Taqman probes and Taq polymerase, the Taqman probe is in parallel with the amplification and the target allyl site of the template DNA. Hybridize to.
  • the extension reaction from the forward primer side reaches the Taqman probe hybridized to the template, the fluorescent substance bound to the 5 ′ end of the Taqman probe is cleaved by the 5 ′ nuclease activity of Taq polymerase. As a result, the released fluorescent substance is not affected by the quencher and generates fluorescence.
  • SNP detection can be performed by measuring the fluorescence intensity.
  • a method combined with a primer extension method can also be mentioned.
  • This method enables high-throughput analysis, and includes the steps of 1) PCR, 2) PCR product purification, 3) primer extension reaction, 4) extension product purification, 5) mass spectrometry, and 6) genotype determination.
  • a primer extension reaction is performed using a genotyping primer designed so that the 3 'end is directly adjacent to the SNP site.
  • the PCR product is denatured at high temperature and excess genotyping primer is added and annealed.
  • ddNTP and DNA polymerase are added to the reaction system and subjected to a thermal cycle reaction, an oligomer that is one base longer than the genotyping primer is generated.
  • the one base-long oligomer generated in this extension reaction differs depending on the allyl depending on the above-described design of the genotyping primer.
  • the purified extension reaction product is subjected to mass spectrometry and analyzed from the mass spectrum.
  • MF20 / 10S is a system that employs this method.
  • complementary and non-complementary primers are used in an ultra-fine region of about 1 femtoliter (1/1000 trillion liter).
  • the translational diffusion time at the level of one molecule of a fluorescent label primer amplified by the conventional PCR method is measured and analyzed.
  • DNA chip method is one of the types that can achieve high throughput.
  • a DNA chip is obtained by aligning and fixing various types of DNA probes on a substrate, and a labeled DNA sample is hybridized on the chip to detect a fluorescent signal from the probe.
  • SNP typing method using a gene amplification method other than the PCR method is the Snapper method.
  • This method is an SNP typing method using an RCA (rolling cycle amplification) method, which is a DNA amplification method in which a circular single-stranded DNA is used as a template and a DNA polymerase synthesizes a complementary strand DNA while moving on the template.
  • the probe is an oligo DNA with a length of 80-90 bases and contains a 10-20 base length sequence complementary to each of the 5 'and 3' ends of the target SNP at both ends, and is annealed to the target DNA and circular Designed to be The probe is designed so that the 3 'end of the probe is a complementary sequence to the target allele.
  • the probe has a backbone sequence with a length of 40-50 bases and includes sequences complementary to two types of RCA amplification primers.
  • Examples of the SNP typing method using a gene amplification method other than the PCR method include a typing method using the UCAN method and the LAMP method.
  • the UCAN method is a method applying the ICAN method, which is a gene isothermal amplification method developed by Takara Bio.
  • a DNA-RNA-DNA chimeric oligonucleotide (DRD) is used as a primer precursor.
  • This DRD primer precursor is designed such that the DNA at the 3 'end is modified so that replication of the template DNA by DNA polymerase does not occur, and the RNA portion binds to the SNP site.
  • the LAMP method is a gene isothermal amplification method developed by Eiken Chemical Co., Ltd. and defines six regions (F3c, F2c, F1c from the 3 ′ end side, B3, B2, B1 from the 5 ′ end side) of the target gene. Amplification is performed using four types of primers (FIP primer, F3 primer, BIP primer, B3 primer) for the six regions. For the purpose of typing, only the target SNP site (one base) is required between F1 and B1, and the FIP primer and the BIP primer are designed so that one base of the SNP comes to the 5 'end.
  • a DNA synthesis reaction occurs from the dumbbell structure, which is the starting structure of the LAMP method, and the amplification reaction proceeds continuously.
  • the DNA synthesis reaction from the dumbbell structure does not occur and the amplification reaction does not proceed.
  • the Invader method is a method using two types of non-fluorescently labeled probes (allele probe, invader probe), one type of fluorescently labeled probe (FRET probe), and cleavase, which is an endonuclease, without using a nucleic acid amplification method.
  • the allele probe has a sequence complementary to the template DNA on the 3 'end side from the SNP site, and has a sequence unrelated to the template DNA called a flap on the 5' side of the probe.
  • the invader probe has a complementary sequence 5 'from the SNP site of the template DNA, and the base corresponding to the SNP site has an arbitrary base.
  • the FRET probe has a sequence complementary to the flap sequence on the 3 'side.
  • One 5 'side is labeled with a fluorescent dye and a quencher, but the FRET probe is designed to form a double strand in the molecule and is usually quenched.
  • the 3 'end (arbitrary base portion) of the invader probe enters the SNP site when the allele probe forms a double strand with the template DNA.
  • the cleavase recognizes the structure invaded by the base and cleaves the flap portion of the allele probe.
  • the 3 'end of the flap enters the intramolecular double-stranded portion of the FRET probe.
  • cleavase recognizes a structure in which a flap base has entered the FRET probe, and cleaves the fluorescent dye of the FRET probe. Since the fluorescent dye is separated from the quencher, fluorescence is generated.
  • the allele probe does not match the allele, the specific structure recognized by cleavase is not formed, and the flap is not cut.
  • primers for the detection of allele it is designed to be a primer suitable for the region to be amplified and the typing method. For example, it is preferable that the region can be completely amplified, and the sequence can be designed based on sequences near both ends of the region. Primer design methods are well known in the art, and the primers that can be used in the present embodiment satisfy the conditions that allow specific annealing, for example, the length and base composition that allow specific annealing (melting temperature). ).
  • the length of the region to be amplified is not limited as long as typing is not hindered, and may be appropriately increased or decreased depending on the detection method.
  • an allyl site is included in a part of the region to be amplified, but the position of the site in the region to be amplified is not limited, and may be arranged at an appropriate position according to a detection method (typing method).
  • the positional relationship between the primer and the allyl site can be freely designed according to the detection method, and a partial region of the base sequence containing the allyl to be detected (for example, 50 bases or more and 500 bases in length)
  • the primer can be designed in consideration of the characteristics of the typing method as long as it hybridizes to a length of less than or equal to the length.
  • the length that exhibits the function as a primer is preferably 10 to 100 bases or more, usually 15 to 50 bases, preferably 15 to 30 bases.
  • Tm melting temperature
  • the probe When a probe is used for allyl detection, the probe is designed to recognize the allyl moiety.
  • the allyl site may be recognized at any location in the probe in accordance with the typing method, and may be recognized at the end of the probe depending on the typing method.
  • the allele-detecting polynucleotide is used as a probe, the length of the base sequence complementary to the genomic DNA is usually 15 to 200, preferably 15 to 100 bases, more preferably 15 to 50 bases. May be longer or shorter.
  • Preferred Allyl Detection Method for the Present Embodiment include PCR-SSOP (Sequence Specific Oligonucleotide probe) method using PCR, Luminex method, direct sequencing Method, HLA Calculation method, micro SSP method and the like. Specifically, in the PCR-SSOP method, first, a region containing the allyl of the specimen is amplified by PCR using a biotin-labeled primer. Subsequently, the amplified DNA is converted into single-stranded DNA and specifically bound to a probe that is a specific sequence.
  • PCR-SSOP Sequence Specific Oligonucleotide probe
  • a probe is fixed to a microbead that is color-coded with a fluorescent dye, and a fluorescent signal is obtained by binding of fluorescently labeled streptadipine via biotin from the microbead to which the amplified DNA is bound.
  • a fluorescent signal is obtained by binding of fluorescently labeled streptadipine via biotin from the microbead to which the amplified DNA is bound.
  • the gene type can be determined from the type of beads to which the amplified DNA is bound.
  • a commercially available kit may be used for the method. Examples of the kit include, but are not limited to, xMAP (registered trademark) technology (Luminex) that can discriminate many polymorphisms at once. The outline of the method of this embodiment using the Luminex method and this method is demonstrated below for the detection method of allyl.
  • Polynucleotides used for allyl detection are known based on the base sequence of HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allyl, depending on the type of primer or probe and the applicable detection method. Chemical synthesis may be carried out by the oligonucleotide synthesis method described above, and may be synthesized using a commercially available chemical synthesis apparatus. A person skilled in the art can use a known method based on the base sequence of HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allyl and their complementary strands and the positional information of allyl according to the present embodiment. Can be used to synthesize polynucleotides.
  • a polynucleotide may be modified using a fluorescent dye or a nucleotide derivative modified with biotin or the like, or a fluorescent dye or the like may be bound to the synthesized polynucleotide.
  • a PCR reaction is carried out by allowing the above-mentioned primers and heat-resistant DNA polymerase to act on the genomic DNA prepared from the specimen.
  • the above method is described in “Molecular Cloning, A Laboratory Manual 3rd ed. (Cold Spring Harbor Press (2001)) ”can be easily performed by those skilled in the art.
  • Examples of the PCR reaction conditions of the present embodiment include the following conditions. Denaturation temperature: 90-100 ° C Annealing temperature: 40-70 ° C Elongation temperature: 60-75 ° C Number of cycles described above: about 30 to 50 times
  • the amplification reaction may be performed twice or more using two or more sets of primers.
  • the primer used in each amplification reaction may be designed at the same position, or may be designed inside the position of the primer in the first amplification.
  • the nucleic acid fragment of the region containing the allele base sequence according to the present embodiment can be specifically amplified using the sample genomic DNA as a template.
  • the base sequence may be determined, and the determined base sequence may be compared with the base sequence of the allele according to the present embodiment. Thereby, it can be determined whether the sample has the allyl according to the present embodiment.
  • the purification method of the obtained PCR product is not particularly limited. For example, Wizard SV Gel and PCR clean-UP System (Promega), GENECLEAN (Funakoshi), QIAquick (registered trademark) PCR purification Kits (QIAGEN), ExoSAP-IT (GE Healthcare Bioscience A) -There are a method using a cellulose filter paper, a method using a dialysis tube, and the like.
  • agarose gel electrophoresis is performed, base sequence fragments are excised from the agarose gel, Wizard SV Gel and PCR clean-UP System (Promega), GENECLEAN (Funakoshi), QIAquick (Registered Trademark) Gel Extraction. It can be purified by Kits (QIAGEN), freeze & squeeze method or the like.
  • the sequencing method is not particularly limited, and examples thereof include, but are not limited to, a direct sequencing method capable of determining a sequence without cloning an amplified nucleic acid fragment into a vector.
  • Examples of the sequencing method include a commercially available kit such as CEQTMDTCS Quick Start Kit (BECKMAN), BigDye Terminator Cycle Sequencing Ready Reaction Kit ABI310 (Applied Biosystems).
  • a primer that can specifically determine the base sequence of the allyl-containing region according to this embodiment.
  • the primer set to be used can be designed by a known method.
  • the base sequence is compared with the base sequence of the allele according to the present embodiment.
  • the allyl according to the present embodiment can also be detected at the protein level.
  • the allyl according to this embodiment can be detected by using an antibody capable of specifically recognizing the HLA protein having the allyl according to this embodiment.
  • the antibody can be produced by an immunological method using a peptide consisting of any region of the amino acid sequence of HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele as an antigen.
  • the method for producing the antibody and the method for detecting allyl according to the present embodiment using the antibody are not particularly limited.
  • HLA-DPB1 * 02: 01 allyl is The sample shows that the hepatitis B virus-related disease is resistant to canceration due to the progression of the disease state.
  • HLA-A * 33: 03 allyl indicates that the sample is susceptible to onset of canceration due to the progression of hepatitis B virus-related disease.
  • the method of the present embodiment can also be said to be a method for examining canceration caused by the pathological progression of a chronic hepatitis B virus-related disease using the allyl according to the present embodiment.
  • hepatitis B virus-related diseases in patients with hepatitis B can be used to calculate the diagnostic utility by using alleles susceptible to canceration and alleles resistant to canceration caused by hepatitis B virus-related diseases. It can also be evaluated by analyzing the positive rate of canceration.
  • “Positive rate” related to chronic hepatitis B refers to the proportion of patients who have one or more susceptible alleles related to canceration due to progression of hepatitis B virus-related disease among all patients, or hepatitis B Refers to the percentage of patients who have no or only one resistant allele for canceration due to the progression of viral-related diseases.
  • the susceptibility allele related to canceration due to pathological progression of hepatitis B virus-related disease Since there are 76 patients with a certain HLA-A * 33: 03 and 397 without any, the positive rate is 16.07%. Furthermore, 12 cases have two HLA-DPB1 * 02: 01, which are resistant alleles related to canceration due to progression of hepatitis B virus-related disease, and 120 cases have only one, Since 341 cases have none, the positive rate is 97.46%.
  • the presence or absence of onset susceptibility to canceration due to the progression of hepatitis B virus-related disease is important information not only for chronic patients with hepatitis B but also for non-chronic patients.
  • treatment of chronic hepatitis B It is important information regarding the selection of methods and therapeutic agents and prevention / prevention of chronic hepatitis B.
  • the presence of this HLA-A * 33: 03 allyl may be either homo or hetero. This is because humans have two types of genes derived from father and mother.
  • the inventors of the present invention have identified 370 HBV patient groups (total 740 alleles) and 2281 healthy control groups (allyl) for HLA alleles related to canceration due to progression of hepatitis B virus-related diseases.
  • the odds ratio (OR; Odds Ratio) in the 95% confidence interval for the total count 4582) was determined.
  • the odds ratio is the ratio between the odds of the HBV patient group and the odds of the healthy control group for the human group having a certain allyl combination.
  • the odds of this HBV patient group are determined by the ratio of the number of HBV patients having a specific allele among the number of HBV patients.
  • the odds of this healthy control group are calculated
  • a group of humans having HLA-DPB1 * 02: 01 as a resistant allyl will be described.
  • the odds ratio is 0.53.
  • the odds ratio of 0.53 for humans with HLA-DPB1 * 02: 01 allyl shown in this specific example is lower than the odds ratio of 0.54 for humans with HLA-DPB1 * 04: 02 allyl.
  • the present inventors calculated the odds ratio of whether or not an HBV patient who progressed due to hepatitis B virus-related disease and progressed to chronic hepatitis became cancerous due to subsequent pathological progression.
  • the odds ratio between a patient who has progressed to chronic hepatitis due to a hepatitis B virus-related disease and who has become cancerous and a patient who has not become cancerous is 0.61. This shows stronger resistance than humans with alleles other than HLA-DPB1 * 02: 01 allele.
  • HLA-DPB1 * 02: 01 allyl has strong resistance to canceration after the pathological condition has progressed due to hepatitis B virus-related disease and has progressed to chronic hepatitis.
  • the odds ratio is smaller than 1, it can be said that the hepatitis B virus-related disease is resistant to canceration due to the progression of the disease state.
  • a human having HLA-DPB1 * 02: 01 as a resistant allyl is resistant to canceration due to progression of a hepatitis B virus-related disease.
  • the allyl according to the present embodiment it is possible to determine the onset resistance or susceptibility to canceration caused by the progression of the hepatitis B virus-related disease.
  • the diagnostic agent for detecting the allele is useful as a diagnostic agent for testing canceration due to progression of the disease state of a hepatitis B virus-related disease.
  • the diagnostic agent can also be used to determine the onset resistance or susceptibility to canceration due to the progression of hepatitis B virus-related disease or the progression of the disease.
  • the allyl according to the present embodiment various primers and probes, an antibody capable of specifically binding to the allyl according to the present embodiment, and diagnostic agents used simultaneously when performing SNP typing (for example, In addition to deoxynucleotide triphosphates (dNTPs, DNA polymerases, buffers, etc.), positive controls, etc., they can be combined with other solvents and solutes to make diagnostic agents. For example, distilled water, pH buffer diagnostic agent, salt, protein, surfactant and the like can be combined.
  • the allele-detecting polynucleotide according to this embodiment may be a chimera of DNA and RNA.
  • the allele-detecting polynucleotide according to the present embodiment may be labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin.
  • the diagnostic agent of this embodiment may further contain a reaction reagent such as a buffer, a dNTP mixture, enzymes (polymerase, etc.) constituting the reaction solution.
  • a reaction reagent is a diagnostic agent having a label detectable by an appropriate chemical or physical detection means.
  • a labeling agent used in a measurement method using such a labeling substance for example, a fluorescent substance, an enzyme, a radioisotope, a luminescent substance and the like are used. An ELISA method using an enzyme for labeling is widely used.
  • fluorescent substances fluorescamine, fluorescein isothiocyanate, etc., as enzymes, peroxidase, alkaline phosphatase, malate dehydrase, ⁇ -glucosidase, ⁇ -galactosidase, etc., as radioisotopes, 125I, 131I, 3H, 14C
  • the luminescent substance include luciferin, lucigenin, luminol, luminol derivatives and the like.
  • reaction medium includes a buffer solution that gives the optimal conditions for the reaction or is useful for stabilizing the reaction product, a stabilizer for the reactant, and the like.
  • the diagnostic agent of the present embodiment it is possible to perform the most convenient and efficient measurement for that purpose by making the diagnostic agent of the present embodiment into a kit.
  • the configuration and form of the assay kit are not particularly limited, and the content thereof is not limited as long as the predetermined purpose can be achieved. In general, it is composed of an instruction manual relating to the means for detecting allyl according to the present embodiment, a reaction reagent, a reaction medium in which a reaction is performed, a base material that provides an assay field, and the like.
  • a collation sample, a detector, or the like for use as a comparison reference or for creating a calibration curve may be included.
  • the detection confirmation means for gene introduction include those capable of detecting the above-mentioned label such as a spectroscope, a radiation detector, and a light scattering detector.
  • the above-described method of the present embodiment may be used in combination with an allele related to canceration caused by the progression of other hepatitis B virus-related diseases.
  • the allele used may be allele related to HLA-A * 33: 03 and / or HLA-DPB1 * 02: 01 allele as long as it is related to canceration due to progression of hepatitis B virus-related disease.
  • Allyl in a sequence other than the above sequence may be used.
  • the method of this embodiment is based on the detection of allyl.
  • allyl is defined as a base change occurring at a frequency of 1% or more of the population, and those with less than 1% are called rare variations.
  • the allele in addition to the above allyl, may be less than 1% regardless of the existence frequency of allyl.
  • the allele combined with this embodiment may be present anywhere in the gene related to canceration due to the progression of hepatitis B virus-related disease, exon, intron, 3′-UTR or 5′-UTR. And its adjacent region and promoter region.
  • Such polynucleotides for detecting alleles can be prepared by those skilled in the art using the method described in the above “(2-2) Detection of alleles”.
  • the number of alleles is not particularly limited, and may be any one of substitution, deletion, insertion and addition of 1 to several tens of bases.
  • SNP single nucleotide polymorphism
  • RFLP restriction fragment length polymorphism
  • VNTR variable number of antrepreneurial
  • type of antreprene also good.
  • Allyl may be a known polymorph or a new polymorph.
  • a polymorphism that is a detection target candidate can be selected from publicly known polymorphisms disclosed in public databases such as GenBank. Further, public data: ENSEMBL (http://www.ensembl.org/) can be used for selection or a haplotype can be used. The selection method and typing method of SNPs constituting the haplotype are as described above. The evaluation of whether the haplotype is related to canceration due to the progression of the hepatitis B virus-related disease can be determined by a statistical test as in the Examples.
  • allyl according to this embodiment in combination with other alleles is also preferable in that the reliability of diagnosis can be increased because canceration due to the progression of hepatitis B virus-related disease can be determined more quickly and accurately. .
  • the frequency of mutation in the hepatitis B virus population infected with the subject is determined based on the progression of the pathological condition when suffering from a hepatitis B virus related disease.
  • the method of testing the ease of pathological progression when a subject suffers from a hepatitis B virus-related disease which further includes a step of comparing with a second standard of ease, has been proved effective. .
  • the mutation (change) of the virus population means that the HBV infected with the subject is mutated in the subject's body, whereby the gene sequence of the HBV is mutated, or for some reason as the virus population.
  • the mutation ratio changes.
  • the frequency of mutation refers to the frequency of HBV having a specific gene sequence or amino acid sequence in the body of a subject.
  • HBV100 hepatitis B virus in which the 100th base of a specific gene of HBV is mutated
  • HBV100 hepatitis B virus in which the 100th base of a specific gene of HBV is mutated
  • the number of HBV100-derived genes is larger than the number of HBV-derived genes of other gene sequences.
  • the second criterion is the 166 highest position at the position of the HBs antigen gene (PreS1 / 2-S gene, HBs antigen protein) in the HBV gene sequence derived from the subject. If the frequent amino acid is leucine, the most frequent amino acid at 236 is glutamine, the most frequent amino acid at 251 is arginine at that position, and the most frequent amino acid at 275 at that position is leucine, The subject is more likely to develop the disease state and more likely to become cancerous.
  • the second criterion is that the 166 most frequently occurring amino acid is not leucine at the position of the HBs antigen gene (PreS1 / 2-S gene) in the HBV gene sequence derived from the subject, and 236 most frequently at that position. If the frequent amino acid is not glutamine, the most frequent amino acid at 251 is not arginine at the position, and the most frequent amino acid at 275 is not leucine at the position, the subject is unlikely to develop a pathological condition and is difficult to become cancerous. .
  • the second criterion does not require that the mutations coincide at all the most frequent amino acid positions on the above gene sequences, and only a part may be mutated.
  • the evaluation as to whether the frequency of mutation in the virus population described above is related to canceration due to the progression of the hepatitis B virus-related disease can be determined by a statistical test as in the Examples.
  • the frequency of mutations in the hepatitis B virus population as the second criterion for the ease of progression of the pathological condition when the subject suffers from a hepatitis B virus-related disease, only the subject's HLA type It is possible to predict the ease of progression of the subject's disease state with higher accuracy than predicting the ease of progression of the subject's disease state. If it is possible to accurately predict the ease of progression of the pathological condition of the subject, for example, the period for follow-up of a patient continuously infected with hepatitis B virus can be set more appropriately. Can be reduced and medical costs can be reduced.
  • the search method the combination of the HLA-DPB1 * 02: 01 allele and the mutation of the HBV population has been described.
  • the present invention is not limited to this, and the search is performed by combining other HLA types and mutations of the HBV population.
  • a combination of another HLA type and the second standard can be determined.
  • the gene sequence of hepatitis B virus infecting the subject is analyzed, and the HLA type of the subject, By combining with the gene sequence of hepatitis B virus, it is possible to accurately predict the ease of progression of the disease state of the subject.
  • Hepatitis B virus population comprising a primer set for determining the frequency of mutation in the hepatitis B virus population or a specific binding substance for determining the amino acid sequence of the HBs antigen of hepatitis B virus.
  • the diagnostic agent can also be used to determine the onset resistance or susceptibility to canceration due to the progression of hepatitis B virus-related disease or the progression of the disease state.
  • various primers and probes for measuring the frequency of mutation of the hepatitis B virus population according to the present embodiment antibodies that can specifically bind to a mutant protein derived from hepatitis B virus, and positive In addition to controls, etc., it can be combined with other solvents and solutes to make a diagnostic agent.
  • distilled water, pH buffer diagnostic agent, salt, protein, surfactant and the like can be combined.
  • specific binding substances include antibodies, antibody fragments, aptamers and the like.
  • the antibody can be produced, for example, by immunizing an animal such as a mouse with a mutated hepatitis B virus protein as an antigen. Alternatively, for example, it can be prepared by screening a phage library. Examples of antibody fragments include Fv, Fab, scFv and the like. The above antibody is preferably a monoclonal antibody. A commercially available antibody may also be used.
  • An aptamer is a substance having a specific binding ability to a target substance.
  • examples of aptamers include nucleic acid aptamers and peptide aptamers.
  • a nucleic acid aptamer having a specific binding ability to a target substance can be selected by, for example, a systematic evolution of ligand by exponential enrichment (SELEX) method.
  • Peptide aptamers having specific binding ability to the target substance can be selected by, for example, the two-hybrid method using yeast.
  • Haplotypes related to progression of hepatitis B chronicity The present inventors performed HLA typing on Japanese HBV patient group 1033 samples and healthy control group 942 samples and suffered from hepatitis B Haplotypes that are sensitive to the progression of chronic hepatitis B are HLA-DPB1 * 09: 01, HLA-DRB1 * 15: 02, and HLA-DQB1 * 06: 01
  • HLA-DPB1 * 04 01
  • HLA-DRB1 * 13 02
  • HLA-DQB1 * 06 04 that are resistant to the progression of hepatitis to chronicity.
  • GenBank National genetic sequence database
  • DDBJ DNA Data Bank of Japan
  • IPD- Data registered in a database such as IMGT / HLA (Immno Polymorphism Database; http://www.ebi.ac.uk/ipd/imgt/hla/) may be used.
  • GenBank the base sequence of the cDNA of HLA-DPB1 * 09: 01 is registered as, for example, AY804139, and the amino acid sequence thereof is registered as, for example, AAW78744.
  • GenBank the base sequence of HLA-DRB1 * 15: 02 cDNA is registered as, for example, X64546, and the amino acid sequence thereof is registered as, for example, CAA45844.
  • GenBank registers the nucleotide sequence of HLA-DQB1 * 06: 01 cDNA, for example, AF184882, and the amino acid sequence, for example, AAD56412.
  • GenBank registers the base sequence of HLA-DPB1 * 04: 01 cDNA, for example, AY804136, and the amino acid sequence, for example, AAW78741.
  • GenBank registers the base sequence of HLA-DRB1 * 13: 02 cDNA, for example, U83584, and the amino acid sequence, for example, AAC02813.
  • GenBank registers the base sequence of HLA-DQB1 * 06: 04 cDNA, for example, AY036896, and the amino acid sequence, for example, AAK96012.
  • a method for detecting a predisposition relating to the progression of hepatitis B to chronicity includes the following steps: a) When the subject's HLA type is continuously infected with hepatitis B virus Comparing with a first criterion of ease of pathological progression.
  • haplotypes that are susceptible to the progression of hepatitis B chronicity are HLA-DPB1 * 09: 01, HLA-DRB1 * 15: 02, and HLA- It is a haplotype containing an allyl combination of DQB1 * 06: 01.
  • the haplotype that is resistant to the progression of hepatitis B chronicity includes haplotypes that include combinations of alleles of HLA-DPB1 * 04: 01, HLA-DRB1 * 13: 02, and HLA-DQB1 * 06: 04 It is.
  • the method of the present embodiment is a method for examining whether or not a patient who is persistently infected with the hepatitis B virus using the haplotype according to the present embodiment progresses to chronic hepatitis.
  • the HLA haplotype of each specimen of each specimen of this embodiment is randomly extracted from the population, and the phase of each specimen (combination of HLA haplotypes) is determined so that the probability that the HLA haplotype composition in the sample is generated is the highest. did. More specifically, in the detection of the haplotype of each specimen of this embodiment, it is considered that the HLA haplotype was randomly extracted from a population in the Hardy-Weinberg equilibrium, and the HLA haplotype composition in the sample is obtained by mutation and recombination. The phase of each specimen (combination of HLA haplotypes) was determined so that the probability of occurrence was highest. The haplotype can be detected by the method described above. About the haplotype diagnostic agent of this embodiment, it is the same as that of what was demonstrated in 1st Embodiment mentioned above.
  • the present invention includes (1) a step of collecting a genomic DNA sample from a subject, (2) a step of determining the HLA type of the subject using the genomic DNA sample, and (3) a subject.
  • the examiner's HLA type includes HLA-A * 33: 03
  • the subject comprises a step of diagnosing that he / she is likely to progress to liver cancer when suffering from chronic hepatitis B.
  • a method for diagnosing easiness of progression to liver cancer when a person suffers from chronic hepatitis B is provided.
  • the present invention also includes (1) a step of collecting a genomic DNA sample from the subject, (2) a step of determining the HLA type of the subject using the genomic DNA sample, and (3) the subject's
  • the HLA type includes HLA-DPB1 * 02: 01
  • the subject is diagnosed as having difficulty developing into liver cancer when suffering from chronic hepatitis B, and the subject has B
  • a method for diagnosing the ease of progression to liver cancer when suffering from chronic hepatitis B is provided.
  • (2) described above is a step of determining the HLA type of the subject by a specific method using the genomic DNA sample from the subject.
  • Specific methods include TaqMan PCR method, MALDI-TOF / MS method, ASO (allele-specific oligonucleotide) method, direct sequencing method, RFLP method, invader method, TGGE, DGGE method, MutY enzyme method, microarray, protein transcription It may be at least one of (PTT) method, Snipper method, Luminex method, direct sequence method, HLA Impression method, and micro SSP method.
  • the present invention includes (1) a step of collecting a genomic DNA sample from a subject, (2) a step of determining the HLA type of the subject using the genomic DNA sample, and (3) a subject. If the examiner's HLA type includes HLA-DPB1 * 09: 01, HLA-DRB1 * 15: 02, and HLA-DQB1 * 06: 01, the subject is diagnosed as prone to develop chronic hepatitis B If the subject's HLA type includes HLA-DPB1 * 04: 01, HLA-DRB1 * 13: 02 and HLA-DQB1 * 06: 04, the subject is unlikely to develop chronic hepatitis B A method for diagnosing the ease with which a subject suffering from hepatitis B virus progresses to chronic hepatitis B, comprising the step of diagnosing.
  • (2) described above is a step of determining the HLA type of the subject by a specific method using the genomic DNA sample from the subject.
  • Specific methods include TaqMan PCR method, MALDI-TOF / MS method, ASO (allele-specific oligonucleotide) method, direct sequencing method, RFLP method, invader method, TGGE, DGGE method, MutY enzyme method, microarray, protein transcription It may be at least one of (PTT) method, Snipper method, Luminex method, direct sequence method, HLA Impression method, and micro SSP method.
  • the frequency of mutation in the hepatitis B virus population infected with the subject is determined according to the second of the ease of progression of the pathological condition when suffering from a hepatitis B virus related disease.
  • a step of comparing with a reference may be further included.
  • allyl or haplotype relating to the ease of progression of the disease state when suffering from a hepatitis B virus-related disease will be described by way of examples.
  • the present invention is not limited to these examples, and various modifications are possible. Is included.
  • Example 1 (Allyl detection 1) The purpose of this example was to detect HLA-A * 33: 03.
  • Each sample derived from Japanese was prepared as follows using QIAamp (registered trademark) DNA Mini kit (QIAGEN). First, 20 ⁇ l of QIAGEN Protease was pipetted into a microtube, and then 200 ⁇ l of each sample was added. Further, Buffer AL was added and mixed for 15 seconds, and then incubated at 56 ° C. for 10 minutes to dissolve the sample. The microtube was then spun down for a few seconds to collect the solution on the inside of the lid.
  • QIAamp registered trademark
  • Buffer AL was added and mixed for 15 seconds, and then incubated at 56 ° C. for 10 minutes to dissolve the sample.
  • the microtube was then spun down for a few seconds to collect the solution on the inside of the lid.
  • LABType SSO HLA SSO HLA A Locus kit (Wan Lambda) or WAKFlow (registered trademark) HLA-A typing kit (Yunaga Pharmaceutical) using PCR-SSOP method using the prepared DNA sample. Typing was performed. The experiment was performed according to the instructions, and a multiplex measurement system (Luminex) using xMAP (registered trademark) technology was used. Specifically, 24.5 ⁇ l of amplification reagent and 0.5 ⁇ l of DNA polymerase solution were added to 2 ⁇ l of the above DNA sample, and PCR reaction was performed under the following conditions. Denaturation temperature: 93 ° C.
  • Luminex XYP was set to 37 ° C., and measurement was performed using a template file corresponding to the lot number of the bead mix.
  • the CSV file of the measurement results was opened with WAKFlow (registered trademark) Typing Software, and the positive / negative of each fluorescent bead was automatically determined based on the cutoff value described in the determination table. In the automatic determination, beads having a fluorescence intensity equal to or higher than the cut-off value were positive, beads having a fluorescence intensity equal to or lower than the cut-off value were negative, and the HLA genotype was determined from the positive / negative pattern of each bead.
  • Table 1 shows a comparison of HLA-A allele frequencies in the liver cancer group (hereinafter, the liver cancer group is also referred to as the HCC group), chronic hepatitis B patient group, and asymptomatic carrier group in Japanese.
  • liver cancer is also referred to as HCC (Hepatocellular Carcinoma).
  • HCC Hepatocellular Carcinoma
  • Table 2 shows a comparison of the HLA-DPB1 allele frequency in the Japanese liver cancer group (hereinafter, the liver cancer group is also referred to as the HCC group), the chronic hepatitis B patient group, and the asymptomatic carrier group.
  • HCC group Japanese liver cancer group
  • HLA-DPB1 * 02: 01 allyl has the onset resistance to canceration due to the progression of hepatitis B virus-related disease.
  • Table 3 the present inventors counted alleles from a sample derived from a patient group (with HCC) who progressed to liver cancer in a HBV patient group and a sample derived from a healthy group.
  • Table 3 shows that HLA-DPB1 * 02: 01 allyl shows a very strong association as HLA-DPB1 allyl that is unlikely to cause liver cancer in HBV infection.
  • the inventors of the present invention also included a sample derived from a patient (with HCC) who has progressed to liver cancer among the HBV patient group and a patient who has not progressed to liver cancer among the HBV patient group ( Alleles were counted from samples from the group without HCC).
  • HLA-DPB1 * 02 01 alleles derived from patients who progressed to chronic hepatitis due to hepatitis B virus-related diseases and became cancerous
  • HLA-DPB1 * 02 01 alleles derived from patients who did not become cancerous
  • the odds ratio with the number was 0.61. That is, humans with HLA-DPB1 * 02: 01 allyl were more resistant to canceration than humans with alleles other than HLA-DPB1 * 02: 01 allyl.
  • HLA-DPB1 * 02: 01 allyl has a strong resistance to the progression of hepatic cancer after hepatitis B virus-related disease progresses to chronic hepatitis. .
  • humans with HLA-DPB1 * 02: 01 allele are less likely to become cancerous due to the progression of hepatitis B virus-related disease than humans without HLA-DPB1 * 02: 01 allele. It was done.
  • the odds ratio is smaller than 1, it can be said that the hepatitis B virus-related disease is resistant to canceration due to the progression of the disease state.
  • a human having HLA-DPB1 * 02: 01 allele as a resistant allyl is resistant to canceration due to progression of hepatitis B virus-related disease.
  • HLA-DPB1 * 02: 01 allele showed a more significant association by analyzing in the group that progressed to liver carcinogenesis due to HBV infection.
  • HLA-DPB1 * 02: 01 allele can be said to be an allele resistant to chronic hepatitis B, but HLA-DPB1 * 02: 01 allele is a more relevant resistant allele against liver carcinogenesis in HBV infection. It can be said.
  • the analysis of alleles related to canceration caused by the progression of hepatitis B virus-related disease in HBV patient groups results in the progression of hepatitis B virus-related disease. Elucidation of the molecular mechanism of saponification and identification of drug discovery target candidate molecules. Further, by analyzing the HBV carrier, it is possible to classify the carrier into a group that is likely to become cancerous and a group that is difficult to become cancerous, and it is possible to provide information that is useful for determining subsequent treatment policies. Furthermore, medical costs can be reduced by developing test kits that contain SNPs of other immune-related genes in addition to the alleles.
  • HBV gene sequence The present inventors analyzed an HBV gene sequence derived from a patient with a hepatitis B virus-related disease who carries HLA-DPB1 * 02: 01 allele.
  • the analysis included HBV derived from 11 cases (hereinafter also referred to simply as cases) of hepatitis B virus-related disease patients who have HLA-DPB1 * 02: 01, HBV derived from 11 cases not developing cancer (hereinafter also simply referred to as control) was used.
  • the age and sex of the patient to be analyzed are matched with each other.
  • High antigenicity is a substance that easily causes an immune reaction when the antigen enters the human body.
  • high antigenicity is a substance that humans can easily recognize as a pathogen.
  • the HBs gene region was divided into three regions and the gene was amplified by the PCR method.
  • the three regions are positions 2814 to 256 on the HBs gene sequence, positions 18 to 557 on the HBs gene sequence, and positions 414 to 989 on the HBs gene sequence.
  • the positions 2814 to 256 on the HBV gene sequence include 2814 to the end 3215 and the beginning 1 to 256. This is because the HBV gene has a circular gene structure.
  • the inventors obtained 66 PCR products from HBV samples derived from 22 cases by gene amplification by the PCR method described above.
  • the present inventors performed deep sequence analysis of 66 PCR products amplified by the PCR method using a high-speed sequencer, and obtained the gene sequence of the HBs gene region.
  • GS Junior or Miseq can be used as the high-speed sequencer.
  • the present inventors acquired 1000 or more data for each base on the HBs gene.
  • the present inventors have obtained amino acid mutation patterns in the HBV gene by listing the variations of HBV mutations from each base on the obtained HBs gene.
  • the present inventors compared the appearance order of amino acids for each position on each gene sequence in the HBV gene derived from the case and from the control based on the obtained mutation pattern.
  • the amino acid having the highest appearance rank for each position on each gene sequence in the HBV gene is also described as the most frequently occurring amino acid.
  • amino acids (hereinafter also simply referred to as AA) are represented by single letter amino acids. For example, “L” is “leucine” and “Q” is “glutamine”.
  • the present inventors among patients with hepatitis B virus-related diseases who possess HLA-DPB1 * 02: 01, developed HBV from 11 patients who developed liver cancer, and developed liver cancer.
  • the HBs gene region of the HBV gene was analyzed using HBV derived from 11 cases that were not.
  • HBV / C is a type of hepatitis B virus that is frequently infected by Japanese and Asians. Comparing the amino acid sequence of the HBs protein derived from a patient with a virus-related disease and the amino acid sequence of a general HBV / C HBs protein, the 166th, 236th, 251st and 275th amino acids were mutated. Admitted. Furthermore, the amino acid at the position where mutation was observed was compared between the amino acid sequence of HBs protein derived from 11 cases that developed liver cancer and the amino acid sequence of HBs protein derived from 11 cases that did not develop liver cancer. .
  • the amino acid at the 166th position in the amino acid sequence of the HBs protein is 51.5% for L, 34.3% for L, and 12.5 for N among samples derived from 11 cases not developing liver cancer.
  • %, X is 1.7%, and S is 0%, L is 99.4%, N is 0%, “X” is 0.6% among samples from 11 patients who developed liver cancer Met. Note that “X” is a set of detected amino acids whose appearance frequency is less than the reference value of 5%.
  • the amino acid at position 236 in the amino acid sequence of the HBs protein is P of 40.2%, Q of 39.8%, and L of 19.4 out of samples derived from 11 cases not developing liver cancer. %, “X” is 0.6%, and P is 12.8%, Q is 87.0%, L is 0.0% among samples from 11 patients who developed liver cancer, “X” ”Was 0.2%.
  • the amino acid at position 251 in the amino acid sequence of the HBs protein is L. 58.3%, R is 40.9%, and “X” is 0 among samples from 11 cases not developing liver cancer. Among the samples from 11 cases that developed liver cancer, L was 12.5%, R was 87.1%, and “X” was 0.4%.
  • the amino acid at position 275 of the amino acid sequence of the HBs protein is 57.8% for L, 39.4% for L, and 2 for "X" among samples from 11 cases not developing liver cancer.
  • Q was 12.1%
  • L was 86.9%
  • X was 0.9%.
  • the amino acid at position 310 of the amino acid sequence of the HBs protein is 11.8% for S, 49.5% for T, and 38.3 for E among samples derived from 11 cases not developing liver cancer. %, “X” is 0.3%, and S is 98.7%, T is 0.0%, E is 0.0%, and “X” "Is 1.3%.
  • the present inventors in Table 6 the pathological progress to liver cancer among the 166th, 236th, 251st, 275th, and 310th of the HBV gene shown in Table 5
  • the odds ratio of the risk of developing liver cancer in the case of an amino acid different from the most frequently occurring amino acid derived from the subject was calculated. There were no exceptional cases for the amino acids at positions 166 and 310 of the HBV gene, and the odds ratio could not be calculated.
  • the odds ratios of the 236th, 251st and 275th HBV genes were 0.01, respectively, indicating resistance to canceration.
  • the 166th most frequently occurring amino acid of the gene derived from the hepatitis B virus population is not leucine, it can be said that it is a criterion that it is difficult to progress to liver cancer when suffering from a hepatitis B virus related disease.
  • the 236th most frequently occurring amino acid of the gene derived from the hepatitis B virus population is not glutamine, it can be said that it is a criterion that it is difficult to progress to liver cancer when suffering from a hepatitis B virus related disease.
  • the 251st most frequent amino acid of the gene derived from a hepatitis B virus population is not arginine, it can be said that it is a standard that it is difficult to progress to liver cancer when suffering from a hepatitis B virus related disease.
  • the 275nd most frequently occurring amino acid of the gene derived from the hepatitis B virus population is not leucine, it can be said that it is a criterion that it is difficult to progress to liver cancer when suffering from a hepatitis B virus related disease.
  • HBV HBs protein mutations derived from 11 patients with hepatitis B virus-related diseases who have HLA-DPB1 * 02: 01 the 166th position is mutated to leucine 10.7%, 166th mutated to leucine, 236th to glutamine, 251st to arginine, and 275th to leucine, 83.6%.
  • Table 8 shows HLA-DRB1 * 15: 02 and HLA-DQB1 when the allele of HLA-DPB1 is 09:01 among the haplotypes related to the progression of hepatitis B chronicity.
  • 06:01 it is shown that there is sensitivity to the pathological progression to chronicity of hepatitis B. Specifically, 14.7% of each sample from the HBV patient group and 8.3% of each sample from the healthy control group possessed the sensitive allyl.
  • the odds ratio for HLA-DRB1 * 15: 02 and HLA-DQB1 * 06: 01 was 1.91.
  • the odds ratio is larger than 1, it can be said that it is sensitive to the pathological progression to the chronicity of hepatitis B.
  • HLA-DPB1 * 05: 01 When the odds ratio of HLA-DPB1 * 05: 01 is compared with the odds ratio of HLA-DPB1 * 05: 01, which is an allele having a conventional sensitivity, when HLA-DPB1 is 09:01, HLA-DRB1 * 15: 02 and HLA-DQB1 * 06: 01 were more sensitive.
  • Table 9 shows HLA-DRB1 * 13: 02 and HLA-DQB1 when the allele of HLA-DPB1 is 04:01 among the haplotypes related to the progression of hepatitis B chronicity.
  • 06:04 it is shown that the patient is resistant to progression of hepatitis to chronicity. Specifically, 1.9% of each sample derived from the HBV patient group and 4.1% of 942 healthy control groups possessed the resistant allyl.
  • the odds ratio in the case of HLA-DRB1 * 13: 02 and HLA-DQB1 * 06: 04 was 0.44.
  • the odds ratio is smaller than 1, it can be said that there is resistance to pathological progression to chronicity of hepatitis B.
  • HLA-DPB1 * 13: 02 and HLA-DQB1 * 06: 04 When comparing the odds ratio of HLA-DPB1 * 02: 01 and HLA-DPB1 * 04: 02 with other conventional sensitive alleles, the allele of HLA-DPB1 was 04:01 In some cases, HLA-DRB1 * 13: 02 and HLA-DQB1 * 06: 04 were more resistant.
  • HBV patient groups As described above, among all hepatitis B virus-related diseases, in the case of pathological progression to chronic hepatitis B, alleles related to the pathological progression to chronic hepatitis B are given to HBV patient groups. Analysis makes it possible to elucidate the molecular mechanism of pathological progression to chronic hepatitis B specialized for Asians including Japanese, and to identify drug discovery target candidate molecules. Further, by analyzing the HBV carrier, it is possible to classify the carrier into a group that is likely to become chronic and a group that is difficult to become chronic, and can provide information that is useful for determining a subsequent treatment policy. Furthermore, medical costs can be reduced by developing test kits that contain SNPs of other immune-related genes in addition to the alleles.

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Abstract

Le degré de vulnérabilité d'un sujet à la progression d'un état pathologique, lorsque le sujet est atteint d'une maladie causée par le virus de l'hépatite B, est testé. Le test comprend une étape de comparaison du type HLA du sujet avec une première référence pour déterminer la vulnérabilité à la progression de l'état pathologique lorsque le sujet est atteint de la maladie causée par le virus de l'hépatite B.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020174617A (ja) * 2019-04-22 2020-10-29 ジェネシスヘルスケア株式会社 Ｂ型肝炎及び／又はｃ型肝炎のリスクを判定する方法
JP7138074B2 (ja) 2019-04-22 2022-09-15 ジェネシスヘルスケア株式会社 Ｂ型肝炎及び／又はｃ型肝炎のリスクを判定する方法

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