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WO2019027005A1 - Procédés d'évaluation du risque d'apparition de la tuberculose spécifiquement à une lignée génétique de mycobacterium tuberculosis - Google Patents

Procédés d'évaluation du risque d'apparition de la tuberculose spécifiquement à une lignée génétique de mycobacterium tuberculosis Download PDF

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WO2019027005A1
WO2019027005A1 PCT/JP2018/029060 JP2018029060W WO2019027005A1 WO 2019027005 A1 WO2019027005 A1 WO 2019027005A1 JP 2018029060 W JP2018029060 W JP 2018029060W WO 2019027005 A1 WO2019027005 A1 WO 2019027005A1
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tuberculosis
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徳永　勝士
陽輔 大前
理人 豊岡
英樹 野内
泰誠 莚田
充明 久保
スラカメ マハシリモンコン
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国立大学法人東京大学
国立研究開発法人理化学研究所
タイ国 ミニストリー オブ パブリック ヘルス、デパートメント オブ メディカル サイエンシス
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Publication of WO2019027005A1 publication Critical patent/WO2019027005A1/fr

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6832—Enhancement of hybridisation reaction
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Definitions

  • the present invention relates to a method of determining the risk of developing tuberculosis.
  • Non-Patent Document 1 Identification of human gene polymorphisms associated with the onset of tuberculosis has been variously performed on the entire genome by candidate gene research and the like (see Non-Patent Document 1). On the other hand, researches on gene mutations associated with the onset of M. tuberculosis genome have also been conducted (see Non-patent Document 2). However, there has never been an example in which these human genomes and M. tuberculosis genome were simultaneously analyzed to comprehensively analyze genes involved in the onset of M. tuberculosis gene lineage from the entire genome. This is because it is rare to obtain both human genome information and M. tuberculosis genome information from a patient who has developed a disease.
  • the present invention identifies the human gene associated with tuberculosis onset specifically from the elucidation of the interaction between the human genome and the pathogen genome associated with tuberculosis onset, and in a subject infected with tuberculosis.
  • An object of the present invention is to provide a method for determining the risk of developing tuberculosis specific to each M. tuberculosis strain based on genetic information of a subject.
  • SNP Single Nucleotide Polymorphism
  • the SNP information of the human genome was obtained by DNA extracted from the blood of patients who developed tuberculosis and healthy individuals and a microarray for SNP typing.
  • the genome of infected M. tuberculosis was extracted from the patient and the genetic lineage of M. tuberculosis was identified.
  • principal component analysis was performed using SNPs information of the whole genome in order to make the genetic background of the tuberculosis patient group and the healthy subject group match.
  • tuberculosis patient group according to genetic line of infected tuberculosis bacteria and carry out association analysis to compare allele frequency of each SNPs in patient group infected with tuberculosis common strain and healthy people group
  • SNPs that show a statistical relationship in a genetic line-specific manner of M. tuberculosis have been searched, and the present invention has been completed.
  • the present invention is as follows.
  • [1] In the subject, identify the base of the single nucleotide polymorphism site of the subject's human genome that is associated with the onset of tuberculosis, and type the latent infection of the subject with the T. tuberculosis genetic line, and the subject Method for determining the risk of developing tuberculosis in a Mycobacterium tuberculosis genetic lineage-specifically by Mycobacterium tuberculosis latently infected in [2] The method of [1], wherein the M. tuberculosis genetic line to be typed is selected from the group consisting of Beijing strain, non-Beijing strain, EAI strain and non-EAI strain.
  • a primer for human genome for determining the risk of developing tuberculosis in a M. tuberculosis-infected subject which is a risk of developing tuberculosis in a Beijing strain-infected subject according to any of the following (p1) to (p14):
  • a primer for determining the risk a primer for determining the risk of developing tuberculosis in a non-Beijing strain-infected subject according to any of the following (p15) to (p26), any of the following (p27) to (p34):
  • Primers for determining the risk of developing tuberculosis in any EAI strain infected subject, or for determining the risk of developing tuberculosis in a non-EAI infected subject having any of the following (p35) to (p39)
  • Primer (p1) a primer capable of amplifying a region including the 26th base (a base at a polymorphic site of rs9348878) in the base sequence of S
  • a probe for determining the risk of developing tuberculosis in a T. tuberculosis-infected subject comprising A probe for determining the risk of developing tuberculosis in a Beijing strain-infected subject according to any of the following (q1) to (q14), a non-Beijing strain-infected subject according to any of the following (q15) to (q26): A probe for determining the risk of developing tuberculosis or a probe for determining the risk of developing tuberculosis in a subject infected with any of the EAI strains described in (q27) to (q34) below or (q35) A probe for determining the risk of developing tuberculosis in a non-EAI strain-infected subject of any of (q39): (q1) the 26th base in the base sequence of SEQ ID NO: 1 (base at polymorphic site of rs9348878) A probe capable of hybridizing to a region comprising (q2)
  • kits for determining the risk of developing tuberculosis comprising the primer of [5] or the probe of [6].
  • [8] In a subject, the expression level of a gene located in the vicinity of a single nucleotide polymorphism site of the subject genome which is associated with the onset of tuberculosis is measured, and a latent infection of Mycobacterium tuberculosis A method of typing strains to determine the risk of developing tuberculosis due to a latently infected M. tuberculosis in a subject.
  • tubercle bacillus in which the subject is infected is a non-Beijing strain, it is located in the vicinity of a single nucleotide polymorphism ((nb2) rs1494320) at the 26th base of the nucleotide sequence shown in SEQ ID NO: 16
  • tuberculosis species with which the subject is infected is a non-Beijing strain, it is located in the vicinity of a single nucleotide polymorphism ((nb11) rs6071980) at the 26th base of the nucleotide sequence shown in SEQ ID NO: 25
  • the present specification includes the disclosure content of Japanese Patent Application No. 2017-150296 based on which the priority of the present application is based.
  • a single nucleotide polymorphism is identified, or the expression of a gene adjacent to the single nucleotide polymorphism site is measured, whereby it is possible to use tuberculosis for each genetic line of M. tuberculosis infected with M. tuberculosis. It is possible to determine the risk of onset and efficiently sort out M. tuberculosis-infected persons who are at high risk of onset of tuberculosis from uninfected persons, treat them before onset, and prevent onset.
  • FIG. 7 shows a list of single nucleotide polymorphisms specifically associated with the onset of tuberculosis and genes in the vicinity (specifically, Beijing strain) of M. tuberculosis genetic lineage (the continuation of FIG. 1-1). It is a figure which shows the list
  • FIG. 2 shows a list of single nucleotide polymorphisms specifically associated with the onset of tuberculosis and genes in the vicinity (specifically, non-Beijing strain) of M. tuberculosis genetic lineage (continuation of FIG. 2-1). It is a figure which shows the list
  • the present invention is a method for determining the risk of developing tuberculosis (TB) in a subject based on human and M. tuberculosis genetic information.
  • determining the risk of developing tuberculosis in a subject refers to determining the likelihood or difficulty of developing tuberculosis in a subject.
  • a subject infected with, but not developing Mycobacterium tuberculosis is a subject that is latently infected with Mycobacterium tuberculosis.
  • the present invention is also a method of obtaining ancillary data to determine the risk of developing tuberculosis in a subject.
  • evaluation and determination are also referred to as prediction.
  • Tuberculosis refers to an infectious disease caused by Mycobacterium tuberculosis. The most common site is lung, but it infects organs and organs throughout the body and causes extrapulmonary tuberculosis such as pulmonary tuberculosis or tuberculosis meningitis, tuberculosis lymphadenitis.
  • the risk of developing tuberculosis can be determined, and if it is determined that the risk is high, the onset can be avoided by administering an antibiotic against M. tuberculosis in advance.
  • SNPs single nucleotide polymorphisms
  • analysis of single nucleotide polymorphism means identifying a base at a single nucleotide polymorphism site.
  • the expression of a gene located in the vicinity of a single nucleotide polymorphism site associated with the susceptibility to development of tuberculosis is measured, and the risk of developing tuberculosis is evaluated and determined based on the expression amount.
  • the gene located in the vicinity of the single nucleotide polymorphism site refers to a gene that is close in distance to the single nucleotide polymorphism site, preferably the gene that is the closest in distance.
  • the gene located in the vicinity of the single nucleotide polymorphism site is reduced in expression or enhanced in expression by the allele at the single nucleotide polymorphism site.
  • the risk of developing tuberculosis can be determined using the expression of a gene located in the vicinity of the single nucleotide polymorphism site associated with susceptibility.
  • the genetic lineage (Lineage) of the tubercle bacillus genome shows diversity, such as Beijing (Beijing) strain, EAI (East-African Indian) strain, CAS (Central Asian Strain) strain, Euro-American strain and the like.
  • Beijing strains are often infected, non-Beijing strains are collectively referred to as non-Beijing strains, and non-Beijing strains include EAI strains, CAS strains and Euro-American strains.
  • non-EAI strains include Beijing strains, CAS strains and Euro-American strains.
  • the single nucleotide polymorphisms associated with the risk of developing tuberculosis in a subject are different.
  • the risk of developing tuberculosis varies with the age of the subject, and is referred to as senile or juvenile onset depending on the age.
  • a subject who develops onset is said to be senile onset when the age of the subject is 45 years or older, and that it is juvenile onset when the age is under 45 (Mahasirimongkol S et al. J Hum Genet. 2012 Jun; 57 (6): 363-7 ).
  • Mycobacterium tuberculosis is isolated from a patient who has actually developed tuberculosis, and the genetic lineage of Mycobacterium tuberculosis is determined by typing, and single nucleotide polymorphisms across the patient's genome are analyzed.
  • patients collected from before the association analysis are compared with a group of patients having a genetic background similar to the group of healthy people. It becomes possible to extract. Also, by considering the onset age information of the patients, it is possible to extract a group of patients considered to have a common onset mechanism.
  • a group of patients extracted by such a method based on the genome information of infected tuberculosis bacteria, a group of patients infected with Beijing strain, a group of patients infected with non-Beijing strain, a group of patients infected with EAI strain, non-EAI strain infection
  • a group of patients infected with Beijing strain a group of patients infected with non-Beijing strain
  • a group of patients infected with EAI strain non-EAI strain infection
  • the present inventors have found that multiple single nucleotide polymorphisms on the human genome are associated with the onset of tuberculosis by the method of human whole genome analysis (GWAS), and completed the present invention. Is associated with the occurrence of a single nucleotide polymorphism of allyl and the risk of developing tuberculosis statistically related.
  • GWAS human whole genome analysis
  • Analysis of single nucleotide polymorphisms refers to determining the type of base at single nucleotide polymorphism site, ie, allyl, whether it is detected for one chromosome on a pair of chromosomes or for both chromosomes. Inclusion, detection for both chromosomes also includes detection of homozygosity or heterozygosity at the single nucleotide polymorphism site. Each allele that is opposite to a particular allele contained in a sample isolated from a subject can be detected and genotyped. When only one allyl is detected, it is homozygous having the allele homozygously, and when two allyls are detected, it is heterozygous having the two alleles heterozygously.
  • the risk of developing tuberculosis in a subject with latent infection with M. tuberculosis is determined by the following method.
  • Mycobacterium tuberculosis is isolated from the site of Mycobacterium tuberculosis infection in the subject. Usually, it may be isolated from the subject's sputum.
  • the sputum of M. tuberculosis patient is applied to Lowenstein-Jensen medium and cultured at 37 ° C. for 4 to 6 weeks. Genomic DNA is extracted from the cultured cells.
  • TE 10 mM Tris HCl pH 8.0, 1 mM EDTA
  • kill by heat treatment at 80 ° C. for 20 minutes add 10 mg / ml lysozyme solution, and react overnight Let it be lysed.
  • the isolated M. tuberculosis genomic DNA is typed to determine the genetic lineage. Markers and methods for typing M. tuberculosis are known, and for example, Gagneux S et al. Proc Natl Acad Sci USA A. 2006 Feb 21; 103 (8) which measures the presence or absence of a specific marker by PCR. J. Clin. Microbiol: A method of determining the Large Sequence Polymorphism (LSP) described in 2869-73. Or the DigiTag 2 method developed by the present inventors for SNP typing of the tuberculosis genome (Srilohasin P et al. J. Clin. Microbiol 2014, 52 (6): 1962-8).
  • LSP Large Sequence Polymorphism
  • the LSP determination method when the LSP determination method is classified into EAI strain, Beijing strain, CAS strain and Euro-American strain, RD239, TbD1, RD105, RD750, 7 bp deletion at pks 15 / as a genomic region to be a marker. It uses combining 5 of 1.
  • the presence or absence of the deletion of the target region can be determined by the size of the amplification product, and the genetic line can be determined.
  • EAI strain is RD239 (-) and TbD1 (+)
  • Beijing strain is RD105 (-) and TbD1 (-)
  • CAS strain is RD750 (-) and TbD1 (-)
  • Euro-American strain is TbD1 (-) and 7 bp deletion at pks 15/1.
  • the M. tuberculosis genetic line can be further classified into sublines as well as the above-mentioned classification of genetic lines. It is also possible to further subdivide and determine single nucleotide polymorphisms associated with the onset of tuberculosis for each strain.
  • the genome may be extracted from peripheral blood cells of the subject and single nucleotide polymorphisms may be analyzed, or the expression amount of a gene located in the vicinity of the single nucleotide polymorphism site may be measured.
  • the expression level for example, the expression level in blood may be measured. That is, in the present invention, M. tuberculosis which is latently infected in the subject is collected, and typing is performed to determine the genetic lineage of M. tuberculosis, and at the same time, in relation to the susceptibility to development of tuberculosis in the human genome of the subject.
  • the single nucleotide polymorphism is analyzed, or the expression level of the gene located in the vicinity of the single nucleotide polymorphism is measured.
  • the risk of developing tuberculosis is determined from single nucleotide polymorphism analysis or gene expression level of the subject for each type of finally infected M. tuberculosis.
  • the following single nucleotide polymorphisms are analyzed for a Beijing strain-infected subject, a non-Beijing strain-infected subject, an EAI strain-infected subject, and a non-EAI strain-infected subject, respectively.
  • “RsXXXXXX (X is an arbitrary number)” indicating a single nucleotide polymorphism indicates an rs number which is a reference number of a SNP database (dbSNP BUILD 137) of NCBI (National Center for Biotechnology Information).
  • the following single nucleotide polymorphism is a single nucleotide polymorphism of 1 ⁇ 10 ⁇ 5 or less when it is expressed as P value in relation to the onset of tuberculosis at the time of infection with M. tuberculosis of a specific genetic strain.
  • the P value is a value indicating whether there is a difference in the frequency of single nucleotide polymorphisms between the group of patients who developed tuberculosis and the group of healthy people who do not develop tuberculosis, and the smaller the value, the more likely the correlation is. It is judged.
  • the base at the single nucleotide polymorphism site is represented by a base in either the normal strand or reverse strand sequence registered in the SNP database. In the sequence of the other strand, they are complementary bases.
  • Non-Beijing Strain-Infected Subjects rs12144738, rs1494320, rs1712674, rs1418425, rs4688637, rs12374531, rs11784415, rs2182093, rs10798, rs4267316, rs6071980, rs743057
  • Non-EAI strain infected subject rs1820920, rs11737270, rs10832678, rs10507084, rs1440548
  • the single nucleotide polymorphism represented by the above rs number is a single nucleotide polymorphism described below.
  • odds ratio 95% confidence interval
  • P value P value
  • the odds ratio of a single nucleotide polymorphism is less than 1, a subject having minor alleles in the single nucleotide polymorphism is less likely to develop tuberculosis. For example, at 0.61, the probability of developing tuberculosis decreases 0.61 times.
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 1 is G or A.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of A. That is, it can be determined that a subject carrying G allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying A allele.
  • the odds ratio is 1.98 (1.48-2.64) and the P value is 2.69 ⁇ 10 -6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 2 is A or G.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is A
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of G. That is, it can be determined that the risk of developing tuberculosis at a younger age (less than 45 years old) is higher in a subject carrying A allele (minor allele) than a subject carrying G allele.
  • the odds ratio is 2.00 (1.50-2.67) and the P value is 2.07 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 3 is C or T.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at any age is significantly greater than in the case of T. That is, it can be determined that in subjects carrying C allele (minor allele) there is a higher risk of developing tuberculosis at any age than subjects carrying T allele.
  • the odds ratio is 1.63 (1.32-2.02) and the P value is 5.36 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 4 is C or T.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of T. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis in old age (45 or older) than a subject who holds T allele.
  • the odds ratio is 1.80 (1.39-2.33) and the P value is 6.97 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 5 is C or A.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of A. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject who holds A allele.
  • the odds ratio is 1.95 (1.46-2.60) and the P value is 5.35 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 6 is G or T.
  • the proportion of subjects who develop tuberculosis at any age is significantly smaller than in the case of T. That is, it can be determined that the risk of developing tuberculosis at any age is lower in subjects carrying G allele (minor allele) than subjects carrying T allele.
  • the odds ratio is 0.61 (0.49-0.75) and the P value is 4.31 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 7 is T or C.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is T, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of C. That is, it can be determined that a subject carrying T-allyl (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying C-allyl.
  • the odds ratio is 2.08 (1.50-2.89) and the P value is 7.78 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 8 is T or C.
  • the proportion of subjects who develop tuberculosis at any age is significantly greater than in the case of C. That is, it can be determined that a subject carrying T-allyl (minor allyl) is at a higher risk of developing tuberculosis at any age than a subject carrying C-allyl.
  • the odds ratio is 2.49 (1.70-3.64) and the P value is 1.27 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 9 is A or G.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is A
  • the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of G. That is, it can be determined that a subject who holds A allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than a subject who holds G allele.
  • the odds ratio is 2.31 (1.60-3.35) and the P value is 5.15 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 11 is G or A.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis in the old age (45 years or older) is significantly larger than in the case of A. That is, it can be determined that the subject carrying G allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than the subject carrying A allele.
  • the odds ratio is 2.35 (1.63-3.38) and the P value is 2.97 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 12 is G or A.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is G, the proportion of subjects who develop tuberculosis in the old age (45 years or older) is significantly larger than in the case of A. That is, it can be determined that the subject carrying G allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than the subject carrying A allele.
  • the odds ratio is 2.36 (1.64-3.40) and the P value is 2.02 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 13 is T or G.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is T, the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of G. That is, it can be determined that the subject carrying T allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than the subject carrying G allele.
  • the odds ratio is 2.27 (1.62-3.18) and the P value is 9.83 ⁇ 10 -7 .
  • (b14) rs 1648835 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 14 and is T or G. This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is T, the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of G. That is, it can be determined that the subject carrying T allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than the subject carrying G allele.
  • the odds ratio is 2.33 (1.65-3.29) and the P value is 8.60 ⁇ 10 ⁇ 7 .
  • Non-Beijing Strain-Infected Subjects (nb1) rs12144738 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 15, and is C or T.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of T. That is, it can be determined that a subject carrying C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele.
  • the odds ratio is 2.22 (1.59-3.10) and the P value is 2.08 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 16 is C or T.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of T. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis in old age (45 or older) than a subject who holds T allele.
  • the odds ratio is 1.71 (1.40-1.08) and the P value is 7.84 ⁇ 10 ⁇ 8 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 17 is G or T.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis in the old age (45 years or older) is significantly larger than in the case of T. That is, it can be determined that a subject carrying G allele (minor allele) has a higher risk of developing tuberculosis in the old age (45 years or older) than a subject carrying T allele.
  • the odds ratio is 1.77 (1.40-2.25) and the P value is 1.63 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 18 is T or C.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is T, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of C. That is, it can be determined that a subject carrying T-allyl (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying C-allyl.
  • the odds ratio is 1.74 (1.43-2.12) and the P value is 2.54 ⁇ 10 ⁇ 8 .
  • nb5 rs4688637 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 19 and is C or A.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of A. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject who holds A allele.
  • the odds ratio is 2.08 (1.50-2.88) and the P value is 7.15 ⁇ 10 ⁇ 6 .
  • (nb6) rs12374531 The polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 20, which is G or A.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis in the old age of tuberculosis is significantly smaller than in the case of A. That is, it can be determined that the subject carrying G allele (minor allele) has a lower risk of developing tuberculosis in old age (45 years old or older) than the subject carrying A allele.
  • the odds ratio is 0.58 (0.45-0.74) and the P value is 8.54 ⁇ 10 ⁇ 6 .
  • rs11784415 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 21 and is C or A. This single nucleotide polymorphism is highly associated with juvenile onset. When the base at the single nucleotide polymorphism site is C, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of A. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject who holds A allele. The odds ratio is 2.03 (1.52 to 2.74) and the P value is 2.54 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 22 is T or C.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of C. That is, it can be determined that a subject carrying T-allyl (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying C-allyl.
  • the odds ratio is 2.38 (1.65 to 4.43) and the P value is 1.80 ⁇ 10 -6 .
  • (nb9) rs10798 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 23, and is A or G.
  • the base at the single nucleotide polymorphism site is A
  • the proportion of subjects who develop tuberculosis at any age is significantly greater than in the case of G. That is, it can be determined that a subject who carries A allele (minor allele) is at higher risk of developing tuberculosis at any age than a subject who carries G allele.
  • the odds ratio is 1.60 (1.32-1.95) and the P value is 2.31 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 24 is C or T.
  • the proportion of subjects who develop tuberculosis at any age is significantly greater than in the case of T. That is, it can be determined that in subjects carrying C allele (minor allele) there is a higher risk of developing tuberculosis at any age than subjects carrying T allele.
  • the odds ratio is 2.26 (1.58-32) and the P value is 5.29 ⁇ 10 ⁇ 6 .
  • (nb11) rs6071980 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 25 and is C or T.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of T. That is, it can be determined that a subject carrying C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele.
  • the odds ratio is 2.09 (1.51-2.90) and the P value is 6.77 ⁇ 10 ⁇ 6 .
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 26 is A or G.
  • the proportion of subjects who develop tuberculosis at any age is significantly smaller than in the case of G. That is, it can be determined that the risk of developing tuberculosis at any age is lower in subjects carrying A allele (minor allele) than in subjects carrying G allele.
  • the odds ratio is 0.59 (0.47-0.74) and the P value is 5.70 ⁇ 10 ⁇ 6 .
  • EAI strain infected subject (e1) rs1178938 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 27 and is C or A.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of A. That is, it can be determined that a subject who holds C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject who holds A allele.
  • the odds ratio is 2.37 (1.67-3.35) and the P value is 5.97 ⁇ 10 -7 .
  • the polymorphism at the 26th base of the base sequence shown in SEQ ID NO: 28 is C or T.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of T. That is, it can be determined that a subject carrying C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele.
  • the odds ratio is 2.33 (1.65-3.30) and the P value is 9.72 ⁇ 10 ⁇ 7 .
  • (e3) rs1372667 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 29, and is G or T.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly larger than in the case of T. That is, it can be determined that a subject carrying G allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele.
  • the odds ratio is 2.29 (1.59-3.32) and the P value is 6.43 ⁇ 10 ⁇ 6 .
  • (e4) rs13174549 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 30, and is T or C.
  • T the base at the single nucleotide polymorphism site
  • the odds ratio is 0.44 (0.31-0.62) and the P value is 2.02 ⁇ 10 ⁇ 6 .
  • rs7087410 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 31, and is C or T. This single nucleotide polymorphism is highly associated with juvenile onset. When the base at the single nucleotide polymorphism site is C, the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of T. That is, it can be determined that a subject carrying C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele. The odds ratio is 2.27 (1.58-3.25) and the P value is 5.94 ⁇ 10 ⁇ 6 .
  • rs10898382 It is a polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 32, and is A or C. This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is A
  • the proportion of subjects who develop tuberculosis in old age (45 years or older) is significantly larger than in the case of C. That is, it can be determined that a subject who holds A allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than a subject who holds C allele.
  • the odds ratio is 1.62 (1.32-2.00) and the P value is 3.66 ⁇ 10 ⁇ 6 .
  • Non-EAI strain infected subject rs1820920
  • the polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 35 which is C or T.
  • This single nucleotide polymorphism is highly associated with juvenile onset.
  • the base at the single nucleotide polymorphism site is C
  • the proportion of subjects who develop tuberculosis at a young age (less than 45 years old) is significantly greater than in the case of T. That is, it can be determined that a subject carrying C allele (minor allele) has a higher risk of developing tuberculosis at a younger age (less than 45 years old) than a subject carrying T allele.
  • the odds ratio is 2.04 (1.48-2.82) and the P value is 9.18 ⁇ 10 ⁇ 6 .
  • (ne3) rs10832678 The polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 37, which is G or A.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is G
  • the proportion of subjects who develop tuberculosis in the old age (45 years or older) is significantly larger than in the case of A. That is, it can be determined that the subject carrying G allele (minor allele) has a higher risk of developing tuberculosis in old age (45 years or older) than the subject carrying A allele.
  • the odds ratio is 1.66 (1.33-2.07) and the P value is 5.66 ⁇ 10 ⁇ 6 .
  • (ne4) rs10507084 The polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 38, which is T or C.
  • This single nucleotide polymorphism is highly associated with senile onset.
  • the base at the single nucleotide polymorphism site is T, the percentage of subjects who develop tuberculosis in old age (45 years or older) is significantly smaller than in the case of C. That is, it can be determined that the subject carrying T-allyl (minor allele) has a lower risk of developing tuberculosis in old age (45 years old or older) than the subject carrying C-allyl.
  • the odds ratio is 0.58 (0.46-0.73) and the P value is 4.21 ⁇ 10 ⁇ 6 .
  • the Mycobacterium tuberculosis with which the subject is infected is a Beijing strain
  • at least one of (b1) to (b14) above preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13 or 14 bases
  • at least one of (nb1) to (nb12) above preferably 2 when the M. tuberculosis strain with which the subject is infected is a non-Beijing strain.
  • Tuberculosis which identifies a base of at least one, preferably 2, 3, 4, 5, 6, 7 or 8 single nucleotide polymorphic sites of the above (e1) to (e8) and which is infected with the subject
  • the strain is a non-EAI strain
  • at least one, preferably 2, 3, 4 or 5 single nucleotide polymorphism site bases of the above (ne1) to (ne5) are identified, and the types of bases To determine the risk of developing tuberculosis It is possible.
  • expression of a gene located near the single nucleotide polymorphism site in the subject may be measured.
  • expression of the gene is enhanced or diminished, it can be determined that the subject is at high risk of developing tuberculosis.
  • the method of the present invention can be applied to all human groups in the world because the single nucleotide polymorphisms described above do not exist in human groups.
  • analysis of single nucleotide polymorphism and typing of M. tuberculosis are performed simultaneously, but depending on the human population, there may be one type of M. tuberculosis infected, and in such a case, It may be omitted. For example, most Japanese are of the Beijing type.
  • Non-Beijing Strain-Infected Subjects rs12144738 FHAD1 (Forkhead Associated Phosphate binding Domain 1) (Intron) rs1494320 CD53 (Intron) rs1712674 CD53 (23 kbp 3 ') rs1418425 LRIF1 (Ligand dependent nuclear Receptor Interacting Factor 1) (21 kbp 3 ') rs4688637 PTPRG (Protein Tyrosine Phosphatase, Receptor type G) (Intron) rs12374531 PPP2R2B (Protein Phosphatase 2 Regulatory subunit Bbeta) (52 kbp 5 ') rs11784415 LRRC69 (Leucine Rich Repeat Containing 69) (Intron) rs2182093 PLCE1 (Phospholipase C Epsilon 1) (Intron) rs10798 KCNQ1 (
  • EAI strain infected subject rs1178938 C3orf 58 (Chromosome 3 open reading frame 58) (67 kbp 3 ') rs800065 C3orf 58 (Chromosome 3 open reading frame 58) (68 kbp 3 ') rs1372667 CDH12 (Cadherin 12) (31 kbp 5 ') rs13174549 EBF1 (Early B-cell Factor 1) (Intron) rs7087410 LOC220906 (106 kbp 3 ') rs10898382 DLG2 (Discs Large MAGUK scaffold protein 2) (Intron) rs951729 DLG2 (Discs Large MAGUK scaffold protein 2) (Intron) rs1658693 BTG1 (BTG anti-proliferation factor 1) (26 kbp 5 ')
  • Non-EAI strain infected subject rs 1820920 MIR 4790 (MicroRNA 4790) (301 kbp 5 ') rs11737270 FBXW7 (F-box and WD repeat domain containing 7) (254 kbp 3 ') rs10832678 C11orf 58 (Chromosome 11 open reading frame 58) (7.9 kbp 3 ') rs10507084 RMST (Rhabdoyosarcoma 2 associated transcript) (106 kbp 5 ') rs 1440548 LOC284294 (450 kbp 3 ')
  • 1-1 and 1-2 show the rs number of the single nucleotide polymorphism, the number of the human chromosome where the single nucleotide polymorphism exists, minor allele / major allele, genes located in the vicinity of the single nucleotide polymorphism, onset Shows the P value and odds ratio of each tubercle bacillus genetic line whether it is old age or young age.
  • the above single nucleotide polymorphism and the gene located in the vicinity of the single nucleotide polymorphism site can be referred to as a gene marker for determining the risk of developing tuberculosis.
  • a sample may be collected from a subject and single nucleotide polymorphisms may be analyzed for DNA and RNA of the sample. That is, genomic DNA containing single nucleotide polymorphisms may be extracted from a subject, and bases of single nucleotide polymorphism sites of alleles contained in the extracted DNA may be identified.
  • any sample can be used as long as it is a sample containing chromosomal DNA, and examples thereof include blood, skin, oral mucosa, hair, urine, nails, cells and the like. From these samples, nucleic acids such as chromosomes, DNA or RNA may be isolated and analyzed according to a conventional method.
  • the analysis of single nucleotide polymorphism can be performed by a conventional gene polymorphism analysis method.
  • a conventional gene polymorphism analysis method for example, an analysis method by sequence analysis in which the sequence is directly determined by a known method such as the dideoxy method or Maxam-Gilbert method, a probe specific for gene polymorphism or a hybridization using a microarray (DNA chip) on which the probe is immobilized Methods, various methods using primers specific for gene polymorphism, etc., and more specifically, primer extension method (TaqMan (registered trademark) method), PCR-SSCP method, single-strand conformation polymorphism analysis (SSCP; single strand conformation polymorphism), Invader method, single nucleotide primer method, PCR method, NASBA method, LCR method, SDA method, LAMP method, method using restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis Method (DGGE), Method using chemical cleavage of mismatch site
  • DigiTag 2 method may be used in which each single nucleotide polymorphism genotype is converted to an oligo DNA tag and analysis is performed by hybridization with a DNA chip.
  • the DigiTag 2 method is described in Srilohasin P et al. J. Clin. Microbiol. 2014, 52 (6): 1962-8, Nishida N et al., Analytical Biochemistry 346 (2): 281-288, Nishida et al., Analytical Biochemistry. 364 (1): 78-85 and the like.
  • the analysis by sequence analysis can be performed by a conventional method. Specifically, a sequence reaction is performed using a primer set at the position of several tens of bases on the 5 'side of the single nucleotide polymorphism site, and the base of the single nucleotide polymorphism site can be determined from the analysis result it can.
  • the method of using a primer is carried out by amplifying a nucleic acid sample isolated from a subject using a primer corresponding to a part of a gene sequence containing a single nucleotide polymorphism site. That is, for example, a nucleic acid sample isolated from a subject when each primer is used, using a primer completely or almost completely complementary to one allele and a primer completely or almost completely complementary to the other allele.
  • the allele of single nucleotide polymorphism can be identified depending on whether or not is amplified. Alternatively, only a primer corresponding to one of the alleles may be used.
  • the method using a probe consists of an oligonucleotide corresponding to a part of the gene sequence containing a single nucleotide polymorphism site or a complementary sequence thereof, or an oligonucleotide consisting of a sequence capable of hybridizing to these sequences under stringent conditions.
  • the probe can be used for analysis by hybridization of a nucleic acid sample isolated from a subject, or a nucleic acid sample amplified by a known method such as PCR. That is, complete or almost complete (for example, 70% or more sequence identity, preferably 80% or more sequence identity, of the base sequence portion continuous to the target single nucleotide polymorphism site) to one allyl.
  • Each probe using a probe complementary to% or more of sequence identity, particularly preferably having a sequence identity of 95% or more, and a probe completely or almost completely complementary only to the other allele can be identified depending on whether it hybridizes with the nucleic acid sample isolated from the subject or the nucleic acid sample amplified from the subject when it has been. Alternatively, only probes corresponding to one of the alleles may be used.
  • the hybridization conditions may be any conditions sufficient to distinguish single nucleotide polymorphisms, for example, hybridization occurs when the single nucleotide polymorphism site is a specific allele, while hybridization occurs when the other alleles
  • Conditions that do not cause soybeans, such as stringent conditions can be set as appropriate by those skilled in the art.
  • the probe may be used as a DNA chip (microarray) by fixing one end to a substrate. In this case, even if only a probe corresponding to one gene polymorphism site is immobilized, a probe corresponding to a plurality of single nucleotide polymorphism sites may be immobilized on the DNA chip.
  • Probes and primers for detecting single nucleotide polymorphisms can be appropriately designed based on the sequence information of single nucleotide polymorphisms.
  • the probe consists of a nucleotide sequence (preferably, a DNA fragment) consisting of a nucleotide sequence containing a single nucleotide polymorphism site or a nucleotide sequence complementary to the nucleotide sequence or a sequence capable of hybridizing to the sequences under stringent conditions.
  • the number of bases is 5 to 50, preferably 10 to 30, and more preferably 10 to 25.
  • the primer is a primer capable of identifying an allele at a single nucleotide polymorphism site, and is, for example, a sequence capable of amplifying a region having a sequence containing a single nucleotide polymorphism site.
  • These primers may contain a sequence containing a single nucleotide polymorphism site, and may be 3 'and 5' to a region containing a single nucleotide polymorphism site (preferably a region of 40 to 1000 bases in length).
  • the primer is composed of a nucleotide sequence (preferably a DNA fragment) consisting of a nucleotide sequence containing a single nucleotide polymorphism site or a nucleotide sequence complementary to the nucleotide sequence or a sequence capable of hybridizing to these sequences under stringent conditions.
  • a nucleotide sequence preferably a DNA fragment
  • the number of bases is 5 to 50, preferably 10 to 30, and more preferably 15 to 25.
  • the probe or primer may contain several, preferably 1 to 5, more preferably 1 or 2, particularly preferably 1 mismatch in the continuous base sequence including the single nucleotide polymorphism site. Good.
  • the present invention relates to a probe used to analyze the above single nucleotide polymorphism and a primer used to amplify a DNA fragment containing a single nucleotide polymorphism site to analyze the single nucleotide polymorphism (preferably, at least a pair of primer sets) ). It also includes a DNA chip on which a probe is immobilized. Furthermore, a kit for analyzing the above-mentioned single nucleotide polymorphism site containing a probe, a DNA chip, and a primer is also included.
  • the kit may additionally contain a restriction enzyme, a polymerase, nucleoside triphosphate, a nucleic acid labeling molecule, a buffer, an instruction manual for the kit, etc. which are used for analysis of single nucleotide polymorphisms.
  • the following can be mentioned as a probe or a primer used for analysis of a single nucleotide polymorphism site.
  • a primer capable of amplifying a region containing the 26th base (the base of polymorphic site of rs9348878) in the base sequence of SEQ ID NO: 1 (p2) A primer capable of amplifying a region containing the 26th base (a base at the polymorphic site of rs2070600) in the nucleotide sequence of SEQ ID NO: 2 (p3) A primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs401864) in the nucleotide sequence of SEQ ID NO: 3 (p4)
  • a primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs673119) in the nucleotide sequence of SEQ ID NO: 4 (p5) A primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs1321267) in the nucleotide sequence of SEQ ID NO:
  • a primer capable of amplifying a region including the 26th base (base of polymorphic site of rs12144738) in the nucleotide sequence of SEQ ID NO: 15 (p16) A primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs1494320) in the nucleotide sequence of SEQ ID NO: 16 (p17) A primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs1712674) in the nucleotide sequence of SEQ ID NO: 17 (p18)
  • a primer capable of amplifying a region containing the 26th base (a base at the polymorphic site of rs1418425) in the nucleotide sequence of SEQ ID NO: 18 (p19) A primer capable of amplifying a region containing the 26th base (a base of polymorph
  • a primer capable of amplifying a region including the 26th base (base of polymorphic site of rs1178938) in the nucleotide sequence of SEQ ID NO: 27 (p28) A primer capable of amplifying a region containing the 26th base (the base of polymorphic site of rs8000065) in the nucleotide sequence of SEQ ID NO: 28 (p29) A primer capable of amplifying a region including the 26th base (a base at polymorphic site of rs1372667) in the nucleotide sequence of SEQ ID NO: 29 (p30)
  • a primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs13174549) in the nucleotide sequence of SEQ ID NO: 30 (p31) A primer capable of amplifying a region containing the 26th base (the base of polymorphic site of rs)
  • a primer capable of amplifying a region containing the 26th base (the base of polymorphic site of rs1820920) in the nucleotide sequence of SEQ ID NO: 35 A primer capable of amplifying a region including the 26th base (base of polymorphic site of rs11737270) in the nucleotide sequence of SEQ ID NO: 36 (p37) A primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs10832678) in the base sequence of SEQ ID NO: 37 (p38)
  • a primer capable of amplifying a region containing the 26th base (base of polymorphic site of rs10507084) in the nucleotide sequence of SEQ ID NO: 38 A primer capable of amplifying a region containing the 26th base (a base at polymorphic site of rs 144
  • Probes to determine the risk of developing tuberculosis in a Beijing strain-infected subject (q1) A probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs9348878) in the base sequence of SEQ ID NO: 1 (q2) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs2070600) in the base sequence of SEQ ID NO: 2 (q3) A probe capable of hybridizing to a region containing the 26th base (base of polymorphic site of rs401864) in the base sequence of SEQ ID NO: 3 (q4) A probe capable of hybridizing to a region including the 26th base (base of polymorphic site of rs673119) in the base sequence of SEQ ID NO: 4 (q5) a probe capable of hybridizing to a region including the 26th base (base of polymorphic site of rs1321267) in the base sequence of SEQ ID NO: 5 (q6) A probe
  • Probes for determining the risk of developing tuberculosis in non-Beijing strains infected subjects (q15) A probe capable of hybridizing to a region including the 26th base (a base at the polymorphic site of rs12144738) in the nucleotide sequence of SEQ ID NO: 15 (q16) a probe capable of hybridizing to a region containing the 26th base (the base at the polymorphic site of rs1494320) in the nucleotide sequence of SEQ ID NO: 16 (q17) a probe capable of hybridizing to a region including the 26th base (base of polymorphic site of rs1712674) in the base sequence of SEQ ID NO: 17 (q18) A probe capable of hybridizing to a region including the 26th base (a base at polymorphic site of rs1418425) in the base sequence of SEQ ID NO: 18 (q19) A probe capable of hybridizing to a region including the 26th base (a base at polymorphic site of r
  • Probe for determining the risk of developing tuberculosis in an EAI strain infected subject a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs1178938) in the nucleotide sequence of SEQ ID NO: 27 (q28) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs8000065) in the base sequence of SEQ ID NO: 28 (q29) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs1372667) in the base sequence of SEQ ID NO: 29 (q30) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs13174549) in the base sequence of SEQ ID NO: 30 (q31) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs7087410)
  • Probes for determining the risk of developing tuberculosis in non-EAI strain infected subjects (q35) a probe capable of hybridizing to a region including the 26th base (base of polymorphic site of rs1820920) in the nucleotide sequence of SEQ ID NO: 35 (q36) a probe capable of hybridizing to a region containing the 26th base (the base at the polymorphic site of rs11737270) in the nucleotide sequence of SEQ ID NO: 36 (q37) a probe capable of hybridizing to a region containing the 26th base (the base at the polymorphic site of rs10832678) in the nucleotide sequence of SEQ ID NO: 37 (q38) a probe capable of hybridizing to a region containing the 26th base (the base of polymorphic site of rs10507084) in the base sequence of SEQ ID NO: 38 (q39) a probe capable of hybridizing to a region containing the 26th base (the base of
  • the Mycobacterium tuberculosis with which the subject is infected is a Beijing strain
  • at least one of (p1) to (p14) above preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13 or 14 primers
  • at least one of (p15) to (p26) above preferably 2, when the M.
  • tuberculosis strain with which the subject is infected is a non-Beijing strain
  • 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 primers are used and the tubercle bacillus infecting the subject is the EAI strain
  • the above (p27) to (p34) Using at least one, preferably 2, 3, 4, 5, 6, 7 or 8 primers of the above (p35) when the tubercle bacillus infecting the subject is a non-EAI strain.
  • the single nucleotide polymorphism may be identified using at least one, preferably 2, 3, 4 or 5 primers of to (p39).
  • the primer is preferably used as a primer pair of forward and reverse primers.
  • the M. tuberculosis organism with which the subject is infected is a Beijing strain
  • at least one of the above (q1) to (q14), preferably 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, or 14 probes, and at least one of (q15) to (q26) described above is used, preferably when the M. tuberculosis strain with which the subject is infected is a non-Beijing strain.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 probes are used and the tubercle bacillus infecting the subject is the EAI strain
  • the above (q27) Using at least one, preferably 2, 3, 4, 5, 6, 7 or 8 probes of (q34), when the M. tuberculosis strain with which the subject is infected is a non-EAI strain,
  • a single nucleotide polymorphism may be identified using at least one, preferably 2, 3, 4 or 5 probes of q35) to (q39).
  • the present invention includes a reagent or kit for determining the risk of developing tuberculosis, which comprises the above-described primer or probe.
  • the expression of the gene located in the vicinity of the single nucleotide polymorphism site may be determined by measuring the mRNA of each gene or measuring the expressed protein.
  • mRNA is measured.
  • the mRNA or protein may be measured from tissue or cells.
  • RNA may be extracted from the tissue or cell, reverse transcribed to cDNA, and the cDNA may be measured.
  • the measurement of cDNA can be performed using a probe or a primer that hybridizes complementarily to each gene using sequence information of each gene.
  • the measurement using a probe or a primer can be performed according to the method of detecting the single nucleotide polymorphism described above.
  • the measurement of the protein can be performed by extracting the protein from tissues or cells, and using an antibody such as a monoclonal antibody against the protein by an immunoassay using an antigen-antibody reaction. In addition, it can also be measured by immunohistochemistry, immunocytochemistry staining using the above-described antibody, on collected tissues or cells.
  • the subject is given (1) rifampicin, (2) isoniazid, (3) streptomycin, (4) ethambutol, (5) pyrazinamide, etc.
  • Antibiotics may be combined as appropriate and administered over several months. For example, (1) and (2) and (4) or (3) and (5) may be administered for about 2 months, and thereafter (1) and (2) may be administered for about 4 months.
  • the present invention is based on the analysis results for tuberculosis and healthy people collected in Thailand.
  • the diagnosis of tuberculosis was based on the culture determination of M. tuberculosis from the sputum of the patient, and those that were positive were determined to be those who developed tuberculosis. Patients who were coinfected with the HIV virus are excluded. Healthy persons are those who have no history of tuberculosis. DNA was extracted from the patient's blood and infected M. tuberculosis.
  • the SNP information of the human genome was obtained by Illumina Human610-Quad v1.0 or Illumina Human OmniExpressExome-8 v1.2, which are microarrays for SNP typing manufactured by Illumina. Genotype information derived from 338, 476 SNPs common to the two arrays is to be analyzed in the process of the present invention. For quality control of sample data, a sample with a genotype determination rate of 98% or more for SNPs in the entire genome was adopted.
  • the genetic strain of M. tuberculosis was identified using the extracted M. tuberculosis genome.
  • the determination was carried out using a large sequence polymorphism (LSP) method based on PCR using the extracted genomic DNA as a template.
  • LSP large sequence polymorphism
  • EAI strain is RD239 (-) and TbD1 (+)
  • Beijing strain is RD105 (-) and TbD1 (-)
  • CAS strain is RD750 (-) and TbD1 (-)
  • Euro-American strain is TbD1 (-) and 7 bp deletion at pks 15/1.
  • the other strains were TbD1 (-) and all other four regions (+).
  • Classification was also performed by spoligotyping, but it was consistent with the classification result by the LSP method.
  • non-Beijing strains are collectively referred to as non-Beijing strains, and non-Beijing strains are classified as EAI strains, CAS strains and Euro-American strains. Is included.
  • EAI strain is frequently infected
  • strains other than the EAI strain are collectively referred to as non-EAI strains, and non-EAI strains include Beijing strains, CAS strains and Euro-American strains.
  • a combination of classification of patient groups using patient onset age information is combined to analyze a group of patients considered to have a common onset mechanism, and is unique at the genetic lineage of M. tuberculosis and at a specific onset age
  • the patients were classified according to their age of onset according to the previous report (Mahasirimongkol S et al. J Hum Genet. 2012 Jun; 57 (6): 363-7) according to age group older than 45 and younger group younger than 45 years.
  • the genetic strains of M. tuberculosis found by this search specifically include SNPs associated with the onset of tuberculosis and genes in the vicinity thereof (FIG. 1 to 4).
  • chi-square test is performed on counts of alleles between a group of tuberculosis patients and a group of healthy subjects to calculate P values.
  • the genomic inflation factor which is an indicator of genetic background gap between TB patients and healthy people, was within the acceptable range of 1.066.
  • P values lower than 1E-05 are judged to be suggested associated (suggestively associated).
  • the raw data of genotyping was confirmed for all SNPs whose P value was less than 1E-05, and it was confirmed that there was no problem in genotyping.
  • the number of specimens in the group of patients infected with the strain of Beijing stock in the collected population is smaller than that in the group of all patients who do not consider M. tuberculosis genetic line information (the column of Total in the table).
  • Fourteen SNPs were identified for which more statistically significant associations (P values) were found. These SNPs did not find significant association in the non-Beijing strain-infected patient group. Ten genes were found as the nearest genes to these 14 SNPs (in the table, Gene items).
  • the gene expression level is significantly increased in the patient (before the start of treatment) (PTB_00) compared to the healthy person (Control), 2 months after treatment (PTB_02), 12 months (PTB_02) At PTB_12), the expression level was reduced.
  • the gene expression level of the CD53 gene and the MAFB gene is higher in patients with tuberculosis (Pulmonary TB, PTB) than in patients with latent infection (Latent TB, LTB) infected with but not developing tuberculosis. It was FIG.
  • FIG. 5 shows the results of the CD53 gene
  • FIG. 6 shows the results of the MAFB gene.
  • A indicates the expression level at healthy control (Control), patient (before treatment start, PTB_00), 2 months after treatment (PTB_02) and 12 months (PTB_12), and B indicates healthy people (Control)
  • the expression levels in M. tuberculosis-infected patients (latent infection) (LTB) and (tuberculous onset patients) (PTB) are shown.
  • LTB tumor infection
  • PTB tumor tuberculous onset patients
  • the risk of developing tuberculosis can be determined for each genetic strain of Mycobacterium tuberculosis, and used for diagnosing the possibility of developing tuberculosis Can. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

La présente invention concerne un procédé d'évaluation d'un risque d'apparition de la tuberculose sur la base de l'information génétique d'un sujet, ledit sujet étant infecté par Mycobacterium tuberculosis, spécifiquement à chaque lignée génétique de M. tuberculosis. Le procédé comprend : l'identification d'une base au niveau d'un site de polymorphisme à nucléotide unique dans le génome du sujet concernant l'apparition de la tuberculose chez le sujet ; le typage de la lignée génétique de M. tuberculosis infectant de manière latente le sujet ; et ensuite la détermination du risque d'apparition de la tuberculose causé par l'infection latente par M. tuberculosis chez le sujet spécifiquement à la lignée génétique de M. tuberculosis.
PCT/JP2018/029060 2017-08-02 2018-08-02 Procédés d'évaluation du risque d'apparition de la tuberculose spécifiquement à une lignée génétique de mycobacterium tuberculosis WO2019027005A1 (fr)

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Cited By (1)

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JP2020174642A (ja) * 2019-04-23 2020-10-29 ジェネシスヘルスケア株式会社 結核のリスクを判定する方法

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MAHASIRIMONGKOL S. ET AL.: "Genome-wide association studies of tuberculosis in Asians identify distinct at-risk locus for young tuberculosis", JOURNAL OF HUMAN GENETICS, vol. 57, 2012, pages 363 - 367, XP055679670 *
MEYER C. G. ET AL.: "Human genetic factors in pulmonary tuberculosis: Candidate genes and a genome-wide association study", TROPICAL MEDICINE AND INTERNATIONAL HEALTH, vol. 14, no. 2, 2009, pages 41 *
NONAKA HIDEKI ET AL : "Identification of tuberculosis sensitive gene-loci in Japanese and Thai people", THE JAPANESE SOCIETY FOR TUBERCULOSIS , vol. 88, no. 2, 1 January 2013 (2013-01-01), pages 267 *
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OMAE Y. ET AL.: "Pathogen lineage-based genome-wide association study identified CD 53 as susceptible locus in tuberculosis", JOURNAL OF HUMAN GENETICS, vol. 62, 7 September 2017 (2017-09-07), pages 1015 - 1022, XP055679668 *
OMAE, YOSUKE ET AL.: "Non-official translation: Search for genetic factor on host-side causing tuberculosis crisis on the basis of information about pathogenic bacteria genome", PROGRAMS AND ABSTRACTS OF THE 25TH ANNUAL MEETING OF THE JAPANESE SOCIETY FOR HISTOCOMPATIBILITY, 2016 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020174642A (ja) * 2019-04-23 2020-10-29 ジェネシスヘルスケア株式会社 結核のリスクを判定する方法
JP7137519B2 (ja) 2019-04-23 2022-09-14 ジェネシスヘルスケア株式会社 結核のリスクを判定する方法

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