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WO2018124744A1 - Procédé de diagnostic d'une maladie hépatique par analyse métagénomique bactérienne - Google Patents

Procédé de diagnostic d'une maladie hépatique par analyse métagénomique bactérienne Download PDF

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Publication number
WO2018124744A1
WO2018124744A1 PCT/KR2017/015578 KR2017015578W WO2018124744A1 WO 2018124744 A1 WO2018124744 A1 WO 2018124744A1 KR 2017015578 W KR2017015578 W KR 2017015578W WO 2018124744 A1 WO2018124744 A1 WO 2018124744A1
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derived
extracellular vesicles
bacteria
liver
decrease
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PCT/KR2017/015578
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English (en)
Korean (ko)
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김윤근
박태성
윤정환
임상섭
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주식회사 엠디헬스케어
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Priority claimed from KR1020170180044A external-priority patent/KR101940425B1/ko
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Publication of WO2018124744A1 publication Critical patent/WO2018124744A1/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

  • Liver cancer is one of the three major cancers in the liver, competing with stomach cancer and lung cancer for the second highest cancer mortality rate, commonly referred to as Hepatocellular carcinoma (HCC). It affects people in their 40s and 60s, and four times more men than women. Liver cancer is more likely to occur if you have an underlying liver disease. If you have cirrhosis of any cause, it is a risk factor for liver cancer. Liver cirrhosis occurs in 75-85% of liver cancer patients, and 10-30% of cirrhosis patients develop liver cancer. Viral hepatitis is also an important risk factor. Among them, hepatitis B is involved in 60-70% of liver cancer patients, and hepatitis C is involved in about 10% of liver cancer patients.
  • HCC Hepatocellular carcinoma
  • the present inventors In order to diagnose liver disease based on the causative factors of cirrhosis and liver cancer, the present inventors extracted genes from extracellular vesicles derived from bacteria from blood, a sample derived from a subject, and performed a metagenome analysis on them. Identified bacterial extracellular vesicles that can act as a causative agent of liver disease, and the like, the present invention was completed based on this.
  • the present invention provides a method for providing information for diagnosing liver disease, comprising the following steps.
  • the present invention provides a method for diagnosing liver disease, comprising the following steps.
  • compared to the normal sample derived from the step (c) by comparing the increase or decrease in the content of one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales It may be to diagnose liver cancer.
  • the content of at least one family bacteria-derived extracellular vesicles selected from the group consisting of Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae in comparison to the sample derived from the normal in step (c) It may be to diagnose liver cancer by comparing the increase and decrease.
  • liver cancer may be diagnosed by comparing the increase and decrease of the content of Trabulsiella genus bacteria-derived extracellular vesicles in comparison with the sample derived from the cirrhosis transducer in step (c).
  • the amount of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, and Succiniclasticum is increased in comparison with the sample derived from the normal person in the step (c). It may be to compare the diagnosis of liver cirrhosis.
  • the sample may be blood, and the blood may be whole blood, serum, plasma, or blood monocytes.
  • Extracellular vesicles secreted by the bacteria present in the environment can be absorbed directly into the body and have a direct impact on the development of cancer, liver disease is difficult to diagnose early due to difficult early diagnosis before symptoms appear, so the human-derived according to the present invention Metagenome analysis of bacterial or bacterial-derived extracellular vesicles using a sample predicts the risk of developing liver disease in advance, allowing early diagnosis and prediction of risk groups of liver disease, and delaying the onset or preventing the onset through proper management. In addition, early diagnosis is possible even after the onset of the disease, thereby reducing the incidence of liver disease and increasing the therapeutic effect. In addition, metagenome analysis predicts causative factors in patients diagnosed with hepatic disease, thereby avoiding exposure to causative factors, and improving the progression of liver disease or preventing recurrence.
  • Figure 1 is for evaluating the distribution of bacteria-derived extracellular vesicles in the body
  • Figure 1a after the administration of oral intestinal bacteria (Bacteria) and bacteria-derived vesicles (EV) in the mouth hourly (0, 5min, 3h, 6h, and 12h) is a photograph taken of their distribution
  • Figure 1b is 12 hours after the oral administration of intestinal bacteria (Bacteria) and bacteria-derived extracellular vesicles (EV) to the blood and various organs (heart, Lung, liver, kidney, spleen, adipose tissue, and muscles), and the photographs of the distribution of the bacterial and extracellular vesicles.
  • FIG. 3 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 4 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 5 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles in the liver cancer patients and liver cirrhosis blood.
  • EVs bacteria-derived vesicles
  • FIG. 6 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacterial-derived vesicles from liver cirrhosis cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles from liver cirrhosis cancer patients and normal blood.
  • EVs bacteria-derived vesicles
  • the present invention relates to a method for diagnosing liver disease through bacterial metagenomic analysis, and the present inventors extracted a gene from bacterial extracellular vesicles using a sample derived from a subject and performed a metagenome analysis on the liver disease. Bacterial-derived extracellular vesicles that could act as causative factors were identified.
  • the present invention comprises the steps of (a) extracting DNA from the extracellular vesicles isolated from the subject sample;
  • an information providing method for diagnosing liver disease comprising comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
  • the term "diagnostic liver cancer” means to determine whether there is a possibility of developing liver cancer, relatively high chance of developing liver cancer, or whether liver cancer has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing liver cancer for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of liver cancer and selecting the most appropriate treatment regimen.
  • the term "diagnosing cirrhosis” means to determine whether there is a possibility of developing cirrhosis, a relatively high chance of developing cirrhosis, or whether cirrhosis has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing cirrhosis for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of cirrhosis and selecting the most appropriate treatment regimen.
  • metagenome used in the present invention, also referred to as “metagenome”, refers to the total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, It is mainly used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms which are not cultured.
  • metagenome does not refer to one species of genome or genome, but refers to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • metagenome analysis was preferably performed using bacterial-derived extracellular vesicles isolated from serum.
  • the subject sample may be blood, and the blood may preferably be whole blood, serum, plasma, or blood monocytes, but is not limited thereto.
  • metagenome analysis was performed on extracellular vesicles derived from bacteria in the blood of normal people, liver cirrhosis patients, and liver cancer patients, and were classified into phylum, class, order, and family. Analyzes at the,, and genus levels were respectively identified to identify bacterial-derived vesicles that could actually cause the development of liver cancer and cirrhosis.
  • liver cancer patients as a result of analyzing the bacteria-derived metagenome at the genus level, there is a significant difference between liver cancer patients and normal vesicles derived from Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella (See Example 4).
  • metagenome analysis of extracellular vesicles derived from bacteria in the blood of liver cirrhosis and liver cancer patients was carried out, including phylum, class, order, family, Analyzes at the genus and genus levels, respectively, identified bacterial vesicles that can actually cause liver cancer in cirrhosis transducers.
  • liver-derived liver cancer patients As a result of analyzing the bacteria-derived metagenome at the genus level, there was a significant difference between the liver-derived liver cancer patients and liver cirrhosis vesicles derived from Trabulsiella genus (see Example 5).
  • the metagenome analysis of extracellular vesicles derived from bacteria in the blood of normal people and liver cirrhosis was performed, including phylum, class, order, family, and Analyzes at the genus level identified bacterial vesicles that could actually cause liver cancer in asthma patients.
  • the present invention through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from blood by metagenomic analysis to identify bacteria-derived vesicles significantly changed in liver cancer patients compared to normal people and liver cirrhosis Meta-genomic analysis confirmed that liver cancer can be diagnosed by analyzing the increase and decrease of the content of bacterial-derived vesicles at each level.
  • the present invention through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from the blood by performing a genome analysis of bacteria-derived vesicles with significantly changed content in liver cirrhosis compared to normal people , Metagenomic analysis confirmed that liver cirrhosis can be diagnosed by analyzing the increase and decrease of the content of the bacteria-derived vesicles at each level.
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • the fluorescently labeled 50 ⁇ g of bacteria and bacteria-derived vesicles were administered in the same manner as above 12 hours.
  • Blood, Heart, Lung, Liver, Kidney, Spleen, Adipose tissue, and Muscle were extracted from mice.
  • the intestinal bacteria (Bacteria) were not absorbed into each organ, whereas the intestinal bacteria-derived extracellular vesicles (EV) were detected in the tissues, as shown in FIG. And distribution in liver, kidney, spleen, adipose tissue, and muscle.
  • the blood was first placed in a 10 ml tube and centrifuged (3,500 ⁇ g, 10 min, 4 ° C.) to settle the suspended solids to recover only the supernatant and then transferred to a new 10 ml tube. After removing the bacteria and foreign substances from the recovered supernatant using a 0.22 ⁇ m filter, transfer to centripreigugal filters (50 kD) and centrifuged at 1500 xg, 4 °C for 15 minutes to discard the material smaller than 50 kD and 10 ml Concentrated until.
  • centripreigugal filters 50 kD
  • PCR was performed using the 16S rDNA primer shown in Table 1 to amplify the gene and perform sequencing (Illumina MiSeq sequencer). Output the result as a Standard Flowgram Format (SFF) file, convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and window (20 bps) The part with the average base call accuracy of less than 99% (Phred score ⁇ 20) was removed.
  • SFF Standard Flowgram Format
  • the Operational Taxonomy Unit performed UCLUST and USEARCH for clustering according to sequence similarity. Specifically, the clustering is based on 94% genus, 90% family, 85% order, 80% class, and 75% sequence similarity. OTU's door, river, neck, family and genus level classifications were performed, and bacteria with greater than 97% sequence similarity were analyzed using BLASTN and GreenGenes' 16S DNA sequence database (108,453 sequences) (QIIME).
  • metagenome sequencing was performed after separating vesicles from the blood of 86 liver cancer patients and 331 normal humans with age and gender matching.
  • the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • the diagnostic performance of liver cancer is improved when developing a diagnostic model with one or more biomarkers in bacteria of Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella. Significantly (see Table 4 and FIG. 4).
  • Example 5 Separated from liver cirrhosis and liver cancer patients blood Germ-derived parcel Metagenome Analysis-based Liver Cancer Diagnosis Model
  • Example 3 By the method of Example 3, vesicles were isolated from blood of 84 liver cancer patients and 86 cirrhosis transducers, and metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Cirrhosis Liver cancer t-test Training set Test set Taxon Mean SD Mean SD p-value Ratio AUC sensitivity specificity AUC sensitivity specificity g__Trabulsiella 0.0197 0.0325 0.0436 0.0436 0.0001 2.22 0.73 0.83 0.47 0.55 0.62 0.42
  • liver cancer and cirrhosis By predicting the risk of liver cancer and cirrhosis in advance through metagenomic analysis on the genes present in bacteria-derived extracellular parcel vesicles using a human-derived sample according to the present invention, early diagnosis and prediction of risk groups of liver disease are made through appropriate management. It can delay the onset or prevent the onset, and early diagnosis even after the occurrence of cirrhosis or liver cancer can reduce the incidence of liver disease and increase the therapeutic effect.

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Abstract

La présente invention concerne un procédé de diagnostic d'une maladie hépatique, telle qu'un cancer du foie et une cirrhose, par analyse métagénomique bactérienne et, plus particulièrement, un procédé de diagnostic du cancer du foie et de la cirrhose par la réalisation d'une analyse métagénomique bactérienne à l'aide d'un échantillon dérivé d'un sujet et par l'analyse d'une augmentation ou d'une diminution de la teneur en une vésicule extracellulaire dérivée d'une bactérie spécifique. Une vésicule extracellulaire sécrétée par une bactérie présente dans l'environnement peut être absorbée dans le corps et influencer directement l'apparition d'une inflammation et d'un cancer et le diagnostic précoce d'une maladie hépatique, telle qu'un cancer du foie et une cirrhose, est difficile avant l'apparition d'un quelconque symptôme, ce qui complique un traitement efficace. Ainsi, par l'intermédiaire de l'analyse métagénomique sur un gène présent dans une vésicule extracellulaire dérivée d'une bactérie à l'aide d'un échantillon dérivé du corps humain selon la présente invention, le risque d'apparition d'un cancer du foie et d'une cirrhose peut être prédit à l'avance, ce qui permet un diagnostic précoce et la prédiction d'un groupe à risque de maladie hépatique et de retarder le moment d'apparition ou de prévenir l'apparition par des soins appropriés et un diagnostic précoce est encore possible même après l'apparition de la cirrhose ou du cancer du foie, ce qui peut abaisser le taux d'incidence de la maladie hépatique et améliorer l'effet de traitement.
PCT/KR2017/015578 2016-12-28 2017-12-27 Procédé de diagnostic d'une maladie hépatique par analyse métagénomique bactérienne WO2018124744A1 (fr)

Applications Claiming Priority (6)

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KR10-2016-0181483 2016-12-28
KR20160181483 2016-12-28
KR20170025000 2017-02-24
KR10-2017-0025000 2017-02-24
KR10-2017-0180044 2017-12-26
KR1020170180044A KR101940425B1 (ko) 2016-12-28 2017-12-26 세균 메타게놈 분석을 통한 간질환 진단 방법

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20110025603A (ko) * 2009-09-04 2011-03-10 주식회사이언메딕스 그람 양성 세균유래 세포밖 소포체 및 이의 용도
US20130121968A1 (en) * 2011-10-03 2013-05-16 Atossa Genetics, Inc. Methods of combining metagenome and the metatranscriptome in multiplex profiles
WO2016099076A1 (fr) * 2014-12-16 2016-06-23 이화여자대학교 산학협력단 Procédé d'identification de pathogènes de maladies infectieuses bactériennes à l'aide de nanovésicules dérivées de bactéries
KR20160110232A (ko) * 2015-03-11 2016-09-21 주식회사 엠디헬스케어 유산균 유래 세포밖 소포체를 유효성분으로 포함하는 염증질환의 예방 또는 치료용 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110025603A (ko) * 2009-09-04 2011-03-10 주식회사이언메딕스 그람 양성 세균유래 세포밖 소포체 및 이의 용도
US20130121968A1 (en) * 2011-10-03 2013-05-16 Atossa Genetics, Inc. Methods of combining metagenome and the metatranscriptome in multiplex profiles
WO2016099076A1 (fr) * 2014-12-16 2016-06-23 이화여자대학교 산학협력단 Procédé d'identification de pathogènes de maladies infectieuses bactériennes à l'aide de nanovésicules dérivées de bactéries
KR20160110232A (ko) * 2015-03-11 2016-09-21 주식회사 엠디헬스케어 유산균 유래 세포밖 소포체를 유효성분으로 포함하는 염증질환의 예방 또는 치료용 조성물

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EZZAT, ASMAA ET AL.: "Metagenomic Study of the Liver Microbiota in Liver Cancer-metagenomic and Metatranscriptomic Analyses of the Hepatocellular Carcinoma- associated Microbial Communities and the Potential Role of Microbial Communities in Liver Cancer", JOURNAL OF GASTROINTESTINAL & DIGESTIVE SYSTEM, vol. 4, no. 5, 2014, pages 1 - 10, XP055516508 *

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