KR101418836B1 - Markers for diagnosing liver disease and its use - Google Patents

Abstract

The present invention discloses markers for diagnosing liver disease and a use thereof, and a marker for diagnosing liver disease based on an amino acid variation existing in core protein C region or preC region of HBV, a diagnostic kit, a kit, a microarray and liver disease A diagnostic method is disclosed. The marker for diagnosing liver disease disclosed herein and the method for detecting the same can be useful for early and effective prediction of susceptibility to hepatocellular carcinoma in patients with HBV infection or for diagnosis of liver cancer.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a marker for diagnosing liver disease,

More particularly, the present invention relates to a biomarker capable of diagnosing or prognosing a liver disease including hepatocellular carcinoma, and a method for detecting the biomarker.

Despite the availability of effective vaccines, more than 350 million people worldwide are chronically infected with HBV (Hepatitis B virus, especially Hepatitis B virus, South Korea is known as an endemic area of HBV) To liver disease such as cirrhosis and hepatocellular carcinoma.

Based on a divergence of more than 8% in the complete genome sequence, HBV strains are divided into eight genomic groups or genotypes, denoted AH. Has been proposed to play an important role in HBV genotype and chronic hepatitis B (CHB) (Lee WM. Hepatitis B virus infection. The New England journal of medicine . 1997; 337 (24): 1733-45; Lee DH, Kim JH, Nam JJ, Kim HR, Shin HR. Epidemiological findings of hepatitis B infection based on 1998 National Health and Nutrition Survey in Korea. Journal of Korean medical science . 2002; 17 (4): 457-62; Norder H, Hammas B, Lofdahl S, Courouce AM, Magnius LO. Comparison of the Amino-Acid-Sequences of 9 Different Serotypes of Hepatitis-B Surface-Antigen and Genomic Classification of the Corresponding Hepatitis-B Virus-Strains. J Gen Virol . 1992; 73: 1201-8; Kidd-Ljunggren K, Miyakawa Y, Kidd AH. Genetic variability in hepatitis B viruses. J Gen Virol . 2002; 83: 1267-80; And Miyakawa Y, Mizokami M. Classifying hepatitis B virus genotypes. Intervirology . 2003; 46 (6): 329-38).

In Korea, genotype C2, which is known to be more viral than genotype B, is abnormally dominant, and relatively frequent mutations are found in basic core promoter (BCP) and major hydrophilic regions (MHR) in Korean patients (Kim H, Jee YM, Song BC, et. al . Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences. J Med Virol . 2007; 79 (3): 212-9; And Yoo BC, Park JW, Kim HJ, Lee DH, Cha YJ, Park SM. Precore and core promoter mutations of hepatitis B virus and hepatitis B antigen-negative chronic hepatitis B in Korea. J Hepatol . 2003; 38 (1): 98-103).

On the other hand, over the past decade, the clinical severity of liver disease, especially HBV variants involved in HCC, has increased. To date, several mutation patterns of HBV, such as nucleotide 1762 (A → T) and 1764 (G → A), in the nucleotide 1896 (G → A) or in the double mutation at the basic core promoter (BCP) Have been extensively studied as HBV mutations associated with clinical severity. Two types of clinical severity related mutations, F141L preS2 mutation and S182 *, leading to immature termination from surface antigen (HBsAg), have recently been found in Korean chronic patients (Muns HS, Lee HJ, Hwang ES, Kook YH , Kim BJ Novel F141L Pre-S2 Mutation in Hepatitis B Virus Increases the Risk of Hepatocellular Carcinoma in Patients with Chronic Genotype C Infections J Virol 2011; 85 (1):... 123-32; and SA Lee KK, Kim H, Kim BJ, Nucleotide change of codon 182 in the surface gene of hepatitis B virus genotype C leading to truncated surface protein associated with progression of liver diseases J Hepatol 2012; 56: 63-9 ).

HBcAg, the protein envelope of the viral core, is 183 residues in length and its N-terminal 149 residues are assembly domains. HBcAg is a major target of host immune response, particularly cytotoxic T lymphocyte attack. Mutations that alter the immunoepitope in this part can lead to the production of immune-evading variants, resulting in the continued maintenance of HBV. In addition, since mutations in the C region can cause simultaneous mutations in HBeAg, the key HBV immunoregulatory protein, these mutations can greatly affect the natural pathway of chronic hepatitis B (CHB) .

Although the association between the frequency of preC / C site mutations and the progression of liver disease is known, the effects on single codon mutations or HBeAg serostatus associated with hepatocellular carcinoma (HCC) are not known. Recently, mutations in tumor suppressor genes or oncogenes such as p53, β-catenin, and Axin1 have been found in tumor tissues and they have been reported to be involved in the development of liver cancer. The frequency of gene mutations is so low that it is not enough to account for the causal relationship between these genetic alterations or the incidence of liver cancer. Therefore, the molecular mechanism of the cause and progress of liver cancer remains a challenge. Recently, we report a case of PIVKA-II (prothrombin induced vitamin K absence-II), AFP-L3 (lens cularis agglutinin-reactive), and GPC3 (glypican-3), which are also called biomarkers DCP (Des-gamma carboxyprothrombin) And the need for additional markers that can effectively increase the diagnostic yield of liver cancer.

Korean Patent Laid-Open Publication No. 2010-0039528 relates to the diagnosis of HBV liver cancer, wherein the sialic acid is an enzyme ST3Gal III, a fucose transferase FUT III, a fucose transferase FUT VII or a β1-3 galactosyltransferase protein or Discloses a method for detecting HBV liver cancer markers by a method for detecting expression of a coding gene.

Korean Patent Laid-Open Publication No. 2009-0052670 discloses a composition for diagnosing hepatocellular carcinoma comprising a substance that measures mRNA levels or protein levels of genes such as ANXA2, G22P1, PPIA, CANX, and CXCL16.

Korean Patent Laid-Open Publication No. 2011-0103016 discloses a single nucleotide polymorphism marker of the TGF? III gene for liver cancer diagnosis.

U.S. Patent Publication No. 2010-0092978 discloses a method of diagnosing liver cancer by judging whether the CDH17 is present or up-regulated.

However, these patent documents do not disclose a method for directly diagnosing liver disease or predicting its prognosis from a mutation of a gene encoding a viral core protein derived from hepatitis virus, and there is a need for development of additional markers.

The present invention provides a method for diagnosing liver disease by identifying characteristic mutations in the core protein C region and preC region of HBV associated with liver disease.

In one embodiment herein, the base sequence is a nucleotide sequence encoding one or more codons corresponding to the 5th, 83rd, 97th, 100th, 182th, or 184th of the core protein C region of the HBV, or the 28th amino acid of the preC region And a polynucleotide selected from the group consisting of polynucleotides of 10 to 200 consecutive nucleotide sequences and complementary polynucleotides thereof,

The amino acid residue number of the core protein preC and C region of the HBV follows the corresponding residue order of the preC and C regions of HBV genotype A to H type. The exemplary HBV core preC regions of Form C and Form A are as set forth in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and the Forms C and A of Form A The amino acid residue sequence of the HBV core C region is as shown in SEQ ID NO: 2 and SEQ ID NO: 6, respectively. According to the residue sequence of SEQ ID NO: 6, the 182nd region of the C region corresponds to 184th. In another exemplary embodiment, the pre-C regions of Forms B and D are as set forth in SEQ ID NOs: 9 and 13, respectively, and the C-regions are as described in SEQ ID NOs: 10 and 14, respectively Same as.

The 5th amino acid mutation of the core protein C region of the HBV is at least one of histidine, leucine or threonine (C-P5H / L / T) in proline; The 83rd amino acid mutation is aspartic acid (C-E83D) in glutamic acid; Wherein the 97th amino acid mutation is at least one of phenylalanine or leucine (C-I97F / L) in isosurine; The 100th amino acid mutation is isosuccinic acid (C-L100I) in leucine; Wherein the 182nd or 184th amino acid mutation is at least one of lysine or termination codon (C-Q182K / *, C-Q184K / *) in glutamine; Wherein the 28th amino acid mutation in the preC region is a mutation from a tryptophan to a termination codon (preC-W28 *).

The present invention also provides a marker for diagnosing liver disease, wherein said hepatocellular carcinoma is derived from HBV gene A or C type.

The present invention also pertains to a nucleic acid encoding the HBV core protein preC region according to the present invention having an amino acid sequence represented by SEQ ID NO: 1, 5, 9 or 13, and the C region having an amino acid sequence represented by SEQ ID NO: 2, 6, 10 or 14 , And a marker for diagnosing liver disease.

The present invention also relates to a method for the preparation of a medicament for the treatment and / or prophylaxis of HBV in which the 5th amino acid of the core protein C region of HBV according to the present invention is at least one of histidine, leucine or threonine (C-P5H / L / T) in proline; Aspartic acid (C-E83D) at the 83rd amino acid glutamic acid; At least one of phenylalanine or leucine (C-I97F / L) at the 97th amino acid isosurine; Isosurishin (C-L100I) from the 100th amino acid in leucine; 182 or 184th amino acid is a lysine or a termination codon (C-Q182K / *, C-Q184K / *) in glutamine; And a probe capable of detecting a polynucleotide comprising a site coding for a codon mutation corresponding to at least one mutation selected from the group consisting of a termination codon (preC-W28 *) in tryptophan, or a 28th amino acid of the preC region A primer capable of amplification, or a probe and a primer, wherein the probe and the primer are used singly or together. The amino acid residue number of the core protein preC and C region of HBV depends on the order of residues of HBV genotype A to H type, and the specific sequence and residue variation of preC and C region according to the type are as mentioned above.

The present invention also provides a composition for diagnosing liver disease, wherein the hepatocellular carcinoma according to the present invention is derived from HBV gene A or C type.

The present invention also provides a composition for the diagnosis of hepatocellular carcinoma, wherein the primer according to the present invention is a primer capable of amplifying each of the mutation sites or two or more mutation sites. In one embodiment, the primer comprises the primers of SEQ ID NOS: 17 and 18 Pair, or a pair of primers of SEQ ID NOS: 19 and 20, is provided.

The present invention also provides a kit for diagnosing liver disease comprising a polynucleotide according to the present invention.

The present invention also provides a kit for the diagnosis of liver disease comprising a composition according to the present invention.

The present invention also provides a microarray for diagnosing liver disease comprising a polynucleotide according to the present invention.

The present invention also relates to a method for the diagnosis and prognosis of hepatocellular carcinoma (HCC), comprising the steps of: i) obtaining a nucleic acid sample from a sample of a patient infected with HBV; ii) the mutation (C-P5H / L / T) of the 5th amino acid of the core protein C region of HBV in the obtained nucleic acid sample, the mutation of the 83rd amino acid (C-E83D) / L), the mutation of the 100th amino acid (C-L100I), the mutation (C-Q182K / *, C-Q184K / *) of the 182th or 184th amino acid or the 28th amino acid of the preC region Determining a nucleotide sequence encoding at least one codon corresponding to SEQ ID NO: 1; And iii) at least one codon mutation corresponding to C-P5H / L / T, C-E83D, C-I97F / L, C-L100I, C-Q182K / * or preC- A method of detecting a marker for diagnosing a liver disease comprising the step of correlating the diagnosis with a diagnosis or prognosis of liver disease. The location of the mutated HBV genotypes is as described above.

The method of the present invention provides a method for detecting liver tissue, liver cells, blood, serum, plasma and urine.

The present application also discloses the use of the method of the present invention for sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization , DASH), PCR extension analysis, or TaqMan technique.

In the present invention, when a marker for diagnosing liver disease having a codon mutation corresponding to the amino acid variation of the C region or the preC region of the core protein of HBV or an assay method for detecting the codon mutation is used, And the usefulness to diagnose liver disease, especially hepatocellular carcinoma.

Figures 1A-1C are graphs showing the distribution and frequency of amino acid variations at 29 amino acids of the preC region and 183 amino acids of HBcAg based on the severity of liver disease; The dark and light colored areas represent MHC class I-restricted and MHC class II restricted HBcAg T-cell epitopes, respectively. Star (★) and triplet (▲) refer to specific mutations that affect HCC and affect HBeAg. The red arrow points to six variations, previously known to be negative for HCC.
Figure 2 (a) compares the frequency of preC / C mutations associated with HCC between HCC and control LC and CH patients. Intramemontal mutation types of MHC class II restricted T-cell epitopes are marked with asterisks (*).
Figure 2 (b) compares the frequency of preC / C mutations affecting HBeAg serum status among patients with two different HBeAg serotypes (-, +). Intramemontal mutation types of MHC class II restricted T-cell epitopes are marked with asterisks (*). The red arrow points to six variations, previously known to be negative for HCC.

Hereinafter, specific details for carrying out the present invention will be described in detail, including definitions of terms used in the specification.

As used herein, the term " diagnosis " is intended to include determining an object for a particular disease or disorder, i.e., the susceptibility of the subject, determining whether an object currently has a particular disease or disorder, Or determining the prognosis (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining a stage of a cancer, or determining the reactivity of a cancer for treatment).

As used herein, the term " diagnostic marker or diagnostic marker " refers to a substance capable of differentiating hepatocellular carcinoma cells from normal cells and indicates a distinctive pattern in hepatocellular carcinoma cells compared to normal cells Organic biomolecules such as nucleic acids (DNA, mRNA), polypeptides, proteins, lipids, glycolipids, glycoproteins and the like. More specifically, the term " diagnostic marker or diagnostic marker " may be embodied in a variation due to mutation or deformation at a particular gene locus, and may include a short nucleic acid sequence surrounding the displaced parental or minisatellite ), And in particular about 8 to 1000 bp, which has an amino acid codon variation mainly in MHC Class II restriction sites of the C region or preC region of the core protein of HBV as in the present invention, Particularly about 20 to 600 bp, more particularly about 20 to 100 bp, even more particularly about 10 to 50 bp in length.

As used herein, the term "C-P5H / L / T" refers to the variation of the amino acid codon when the 5th amino acid of the C region of the HBV core protein is replaced by histidine, leucine or threonine in proline .

As used herein, the term "primer" refers to an oligonucleotide having a sequence complementary to a target target nucleotide or nucleic acid and hybridizing thereto, wherein a mononucleotide is added to the 3 'end of the DNA or RNA polymerase in the presence of a DNA or RNA polymerase, Refers to a sequence functioning as a starting point for synthesis, and includes a peptide nucleic acid primer, a labeled primer, a primer in which a phosphodiester bond of a DNA molecule is modified such as a phosphorothioate, and the like.

As used herein, the term " probe " is an oligonucleotide used to detect a target nucleic acid and includes various derivative forms such as hybridization probes, fluorescent probes, FRET probes, and, by itself or with a primer, Presence, or quantitative analysis.

The term "subject" or "patient" as used herein means any single entity in need of treatment, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects and the like. In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

As used herein, the term "sample or sample" refers to a collection of similar cells from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood component. For example, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may include, for example, preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. The sample or sample of the present invention includes, but is not limited to, tissue, cell, whole blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.) and cell culture supernatant. In one embodiment, a human-derived sample is used, and the sample is a tissue, cell, blood, plasma, serum, lymph, bone marrow fluid, saliva, feces, ocular fluid, semen, spinal fluid, joint fluid, thymus fluid, Amniotic fluid and cell tissue fluid, and most preferably tissue, hepatocyte, blood, serum, plasma and urine.

We have investigated the relationship between amino acid changes in the core protein C region and preC region of HBV and hepatocellular carcinoma, especially hepatocellular carcinoma. In particular, the frequency of mutations within MHC class II restriction sites was high, indicating that the frequency of mutations at this location is related to the stage of liver disease progression.

As used herein, the term " liver disease " refers to a symptom in which the liver function is impaired. For example, viral hepatitis (especially hepatitis mediated by HBV), liver cirrhosis and hepatocellular carcinoma. Even if the pathology is unclear, liver diseases include abnormalities in liver function indicators such as GOT, GPT, and γ-GTP. In one embodiment, the liver disease is hepatocellular carcinoma. In another embodiment, the hepatocellular carcinoma is due to one or more of A to H types, particularly A type, more particularly C type HBV infection.

Hepatocellular carcinoma refers to primary hepatocellular carcinoma originating in the liver. Liver cancer metastasized from other sites do not include hepatocellular carcinoma. Hepatocellular carcinoma accounts for more than 90% of all cancers, and 40 to 80% of patients recur, most often recurring in the liver, but may also appear in the lining of the lungs, the inner wall surrounding the abdominal cavity, and the mediastinum. . Causes include hepatitis B virus, hepatitis C virus, alcoholic epilepsy, metabolic chronic epilepsy, and toxic substances.

In one embodiment herein, the liver disease in which the markers, compositions, kits, or methods herein may be used is hepatocellular carcinoma. In particular, the hepatocellular carcinoma is hepatocellular carcinoma derived from HBV. In addition, wild-type HBV can be classified into type A to type H according to its gene sequence, and in one embodiment, hepatocellular carcinoma infected with type A or type HBV. In another embodiment, hepatocellular carcinoma is infected with type C HBV.

In the case of hepatocellular carcinoma, most of the hepatocellular carcinoma is preceded by cirrhosis. Therefore, hepatocellular carcinoma is the intermediate stage between nonneoplastic regenerating nodule and malignant hepatocellular carcinoma And the like. These nodule lesions are called dysplastic nodules and can be subdivided into low grade dysplastic nodules or high grade dysplastic nodule dysplastic nodules. High-grade dysplastic nodules are thought to be precancerous stages of hepatocellular carcinoma as a result of the observation of microscopic hepatocellular carcinomas in the tissues, and hepatocellular carcinomas of this precancerous stage may also be included here. Hepatocellular carcinomas can be divided into four grades according to the Edmonson grade histopathologically, and these stage carcinomas can also be included here.

HBV is a 3.2-kb circular DNA virus with a partially double-stranded genome. There are four overlapping frames in the genome, from which surface proteins (HBs), core proteins (HBc), polymerase X protein (HBx) is expressed (Seeger, C. et al., 2000). Core proteins are involved in the packaging of HBV pol, free genomic RNA (pgRNA) and other proteins, and are known to play an important role in the HBV life cycle. The assembly of core proteins is an important step in the replication of HBV, as the generation of viruses requires assays for core proteins, viral genomes and other proteins.

preC consists of 29 amino acids at the front of the core and produces an e-antigen. HBeAg is a protein secreted by the virus when HBV is actively proliferating in the host and is antigenic. In general, the presence of HBeAg may play an important role in the treatment of chronic hepatitis B because HBeAg detection can be used to detect HBV infection. However, HBeAg-negative patients do not detect HBeAg.

The core protein consists of 183-185 amino acids with two regions consisting of the N-terminal region required for core assembly and the C-terminus regulating viral replication. Core proteins form the capsid of viruses, and capsids play an essential role in the protection of packaged viruses and host factors.

Here we have found a link between mutations in certain amino acids in the preC / C region and liver disease.

Accordingly, in one embodiment, the present invention provides a method for detecting a nucleotide sequence encoding one or more codons corresponding to the 5th, 83rd, 97th, 100th, 182th, or 184th positions of the core protein C region of HBV or the 28th amino acid of the preC region And a polynucleotide selected from the group consisting of polynucleotides of 10 to 200 consecutive nucleotide sequences and complementary polynucleotides thereof.

The residue numbers follow the sequence of the corresponding residues of HBV genotype A to H type identified so far. When the position of the corresponding residue and the specific residue vary depending on the type of HBV, the amino acid corresponds to the corresponding position and the amino acid corresponding to the position. For example, in the case of HBV gene A, the 83rd codon is the aspartic acid (D). In the case of gene A, 2 amino acids are added to the end of the core, and the preC / C total amino acid sequence is 185, , The Q182K / * (termination) of the gene C type corresponds to Q184K / * in the genotype A. In one embodiment, the amino acid residue number of the core protein preC / C region of the HBV may follow the order of residues, for example, genotype A, B, C or D for convenience.

In other embodiments, residue numbers of type A or C are followed. When the sequence of the preC / C region of HBV type C is followed, the mutation associated with diagnosis of liver disease according to the present invention is that the fifth amino acid mutation of the core protein C region of HBV is histidine, leucine or threonine -P5H / L / T); The 83rd amino acid mutation is aspartic acid (designated C-E83D) in glutamic acid; Wherein the 97th amino acid mutation is at least one of phenylalanine or leucine (designated C-I97F / L) in isoruicin; The 100th amino acid mutation is isosurine (named C-L100I) in lucine; The 182nd amino acid mutation may include at least one of lysine or a termination codon (named C-Q182K / *) in glutamine; The 28th amino acid mutation in the preC region is mutated to a stop codon (named preC-W28 *) in tryptophan.

In another embodiment, when the sequence of residues of preC / C region of HBV A type is followed, the 182nd amino acid mutation corresponds to 184th (C-Q182K / *).

As mentioned above, when there is a variation in the position of a specific residue depending on the type of HBV, and when there is a variation in a specific amino acid depending on the corresponding position or position, it includes an amino acid at that position. For example, when the HBV type B residue sequence is followed, the 83rd amino acid is aspartic acid, and the 83rd amino acid is aspartic acid and the 97th amino acid is phenylanamine , Which in this case includes a variation from penny alanine to lucine.

In one embodiment of the invention, the amino acid residue number of the core protein preC region of the HBV is in accordance with the sequence of residues set forth in SEQ ID NO: 1, 5, 9 or 13, and the amino acid residue number of the C region is set forth in SEQ ID NO: 6, 10 or 14. < / RTI >

The sequence of the preC region of the wild-type HBV disclosed herein can be classified as A to H according to the HBV genotype and encompasses all the preC regions present in this type. In one embodiment, it is derived from genotype A or C type. In another embodiment, the core protein preC region is derived from C type, and the protein sequence may be represented by SEQ ID NO: 1, and the nucleic acid sequence may be represented by SEQ ID NO: 3, for example. In another embodiment, the core protein preC region is derived from type A, the protein sequence may be represented by SEQ ID NO: 5, and the nucleic acid sequence may be represented by SEQ ID NO: 7, for example. In another embodiment, the core protein preC region is derived from type B, the protein sequence may be represented by SEQ ID NO: 9, and the nucleic acid sequence may be represented by SEQ ID NO: 11, for example. In another embodiment, the core protein preC region is of type D origin, the protein sequence may be represented by SEQ ID NO: 13, and the nucleic acid sequence may be represented by SEQ ID NO: 15, for example.

The sequence of the C region of the wild-type HBV core protein disclosed herein can be classified as A to H according to the HBV genotype and covers all of the C regions present in this type. In one embodiment, it is derived from genotype A or C type. In another embodiment, the core protein C region is derived from the C-type, and the protein sequence can be represented by SEQ ID NO: 2, and the nucleic acid sequence can be represented by, for example, the sequence of SEQ ID NO: In another embodiment, the core protein C region is derived from type A, and the protein sequence may be represented by, for example, SEQ ID NO: 6, and the nucleic acid sequence may be represented by, for example, the sequence of SEQ ID NO: In another embodiment, the core protein C region is of type B origin, the protein sequence may be represented by SEQ ID NO: 10, and the nucleic acid sequence may be represented by SEQ ID NO: 12, for example. In another embodiment, the core protein C region is of type D origin, the protein sequence may be represented by SEQ ID NO: 14, and the nucleic acid sequence may be represented by SEQ ID NO: 16, for example.

 In one embodiment, the markers for diagnosing liver disease of the present invention were determined to have a high level of mutation in a sample of the hepatocellular carcinoma patient group as compared with the control group (patients with liver cirrhosis and chronic hepatitis), and thus the liver, chronic hepatitis, Can be used for diagnosis and prognosis of hepatocellular carcinoma. Particularly for early detection of susceptibility to human hepatocellular carcinoma, and to assist in the prognosis, diagnosis and treatment of patients presenting with liver cancer.

As the marker according to the present invention, any one of the listed polynucleotides may be used, but two or more markers simultaneously detecting mutations at two or more positions including one or more mutations at one position may be used in combination.

In one embodiment of the present invention, the specimen used for the detection of the marker of the present invention is at least one of hepatitis, hepatocarcinoma tissue, or liver tissue or hepatocellular carcinoma tissue derived from HBV carrier, whole blood, serum or plasma. In another embodiment, the specimen used in the present invention may be a specimen of a normal control group or a specimen of a specific type of hepatocarcinoma control, as well as a specimen of a specimen requiring discrimination for comparative analysis.

In another embodiment herein, the present invention encompasses the use of one or more of histidine, leucine or threonine (C-P5H / L / T) at proline in the 5th amino acid of the core protein C region of HBV; Aspartic acid (C-E83D) at the 83rd amino acid glutamic acid; At least one of phenylalanine or leucine (C-I97F / L) at the 97th amino acid isosurine; Isosurishin (C-L100I) from the 100th amino acid in leucine; The 182nd amino acid is at least one of lysine or a termination codon (C-Q182K / *) in glutamine; a probe capable of detecting a polynucleotide comprising a site in which the 28th amino acid of the preC region encodes at least one codon mutation corresponding to a mutation from a tryptophan to a termination codon (preC-W28 *), or a primer capable of amplifying the polynucleotide , Or a probe and a primer, wherein the amino acid residue number of the core protein preC and C region of the HBV is in the order of residues of the HBV genotype A to H type, and the specific sequence and residue variation of the preC and C regions according to the type As mentioned above. The specific amino acid residues of the core protein preC region and the C region of the HBV are as described above.

Diagnostic compositions according to the present invention may be provided in immobilized form using a variety of methods known in the art for suitable carriers or supports to enhance the speed and convenience of diagnosis (Antibodies: A Laboratory Manual, Harlow &Lane; Cold Spring Harbor, 1988). Examples of suitable carriers or supports are agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose, polyacrylamide, polysterine, filter paper, ion exchange resin, plastic film, plastic tube, glass , Polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like.

The present invention also relates to a kit for liver cancer diagnosis comprising the marker composition comprising the composition for diagnosing liver cancer or one or more polynucleotides for diagnosis of liver cancer.

The diagnostic kit may include not only polynucleotides according to the present invention, but also reagents necessary for the polymerization reaction, such as primers, probes, dNTPs, polymerases and coloring agents.

In addition, a diagnostic marker according to the present invention includes a polypeptide encoded by the polynucleotide in addition to a polynucleotide comprising a mutation site identified herein as described above.

The markers of the present invention can be detected at the protein or nucleic acid level. Methods for detecting protein or nucleic acid levels are known, for example, the former is an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor or ligand that specifically interacts with the marker Or by reaction with cofactors. Reagents or substances that specifically interact or bind to the markers of the present invention can be used in conjunction with chip-based or nanoparticles.

Detection at the nucleic acid level can be carried out by conventional methods such as hybridization using a chip method, polymerase chain reaction using a primer or a probe, and Southern blotting. The detection at the mRNA level is performed using a reverse transcription polymerase chain reaction Reaction, RNase protection assay, or Northern blot. In the present invention, detection includes quantitative and qualitative analysis, including detection of presence or absence, or detection of the amount of expression, and such methods are well known in the art, and those skilled in the art can select an appropriate one for the practice of the present invention There will be.

In one embodiment, PCR (Polymerase Chain Reaction) is used. After amplifying the polynucleotide in the region containing the mutation site, the mutation contained in the amplification product can be detected by base sequence analysis or hybridization. PCR is a method of isolating DNA of a specimen and then detecting a specific gene in a specimen by using a specific primer or a combination of a primer and a probe to determine the presence / absence or the expression level of a specific gene. Such a method is described in, for example, Han, H., Bearss, DJ; Browne, LW; Calaluce, R .; Nagle, RB; Von Hoff, DD. Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002 , 62, (10), 2890-6).

The " primer " which may be included in the compositions or kits of the present application is to be understood as meaning a primer that can hybridize under appropriate conditions in suitable buffers (for example, four different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) Refers to single stranded oligonucleotides that can serve as a starting point for template-dependent DNA synthesis. The appropriate length of the primer may vary depending on the purpose of use, but is usually 10 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. For example, the primer hybridizes to a target DNA containing a displacement site and starts amplification. This primer is used in pairs with a second primer that hybridizes to the opposite side. Amplification amplifies the product from the two primers, indicating the presence of mutations. In another example, the primer may be a pair of primers that bind to both sites including the displacement site.

Primers used in PCR are used in pairs, and primers capable of amplifying mutations according to the present invention, respectively, or multiple, for example, two or more mutations, with a pair of primers can be used. Primers that can amplify each or several mutations at one time can be performed in a single PCR or multiplex PCR manner. Reverse transcription polymerase chain reaction is not significantly different from general PCR method except that cDNA is synthesized from mRNA of a sample for use as a template.

In other embodiments, probes with the primers described above may be used in PCR, for example, using commercially available probes, such as TaqMan (Applied Biosystems, USA) probes, with primers to confirm the mutation . For example, the probe comprises a sequence capable of specifically hybridizing with a mutation site according to the present invention, wherein the primer comprises a peripheral sequence thereof. Whether or not signal amplification (emission) is determined depending on hybridization with the mutation site of the probe can determine whether the used sample is mutated at a specific position.

Other embodiments may be practiced using antibody molecules that specifically bind to the mutated HBV core protein, which is the expression product of a diagnostic marker according to the present invention. The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) Or phage antibody library methods (Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with an antibody-producing lymphocyte, and the techniques required for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay, capture-ELISA, inhibition or competitive assay, sandwich assay, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984, which is incorporated herein by reference.

(C-P5H / L / T), the 83rd amino acid mutation (C-E83D), the 97th amino acid mutation (C-I97F / L ), The 100th amino acid mutation (C-L100I), the 182nd amino acid mutation (C-Q182K / *), or the 28th amino acid mutation (preC-W28 *) in the amino acid sequence of the preC region The present invention relates to a diagnostic kit for liver cancer.

The diagnostic kit of the present invention is as mentioned above with respect to the detection of the presence / absence of the marker, and the marker of the present invention may include reagents necessary for detection at the protein or nucleic acid level. For example, reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands or cofactors, and the like. These reagents can be incorporated into nanoparticles or chips as needed. Reagents that can be detected at the nucleic acid level are reagents used for polymerase chain reaction, reverse transcription polymerase chain reaction, RNase protection assay, northern blot, Southern blot, etc., and include, for example, primers, probes, will be.

The substance that specifically recognizes such a marker of the present invention may be present separately in a divided container, and in this sense, the present invention also encompasses a molecule capable of specifically recognizing the marker of the present invention / RTI >

The specimen to be used with the kit of the present invention is at least one of hepatocellular carcinoma tissue, liver tissue derived from HBV carrier, hepatocellular carcinoma tissue, whole blood, serum or plasma. In another embodiment, the specimen used in the present invention may be a specimen of a normal control group or a specimen of a specific type of hepatocarcinoma control, as well as a specimen of a specimen requiring discrimination for comparative analysis.

In another embodiment herein, the present invention encompasses the use of one or more of histidine, leucine or threonine (C-P5H / L / T) at proline in the 5th amino acid of the core protein C region of HBV; Aspartic acid (C-E83D) at the 83rd amino acid glutamic acid; At least one of phenylalanine or leucine (C-I97F / L) at the 97th amino acid isosurine; Isosurishin (C-L100I) from the 100th amino acid in leucine; 182 or 184th amino acid is a lysine or a termination codon (C-Q182K / *) in glutamine; wherein the 28th amino acid of the preC region encodes at least one codon mutation corresponding to a mutation from tryptophan to a termination codon (preC-W28 *).

The microarray (or chip based assay) may comprise DNA or RNA polynucleotides. The microarray is a probe, and a conventional microarray is used except that it contains the polynucleotide of the present invention. Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The term " probe polynucleotide " means a polynucleotide capable of hybridizing with a corresponding polynucleotide in a specimen, and means an oligonucleotide capable of sequence-specific binding to the complementary strand of the nucleic acid. Such probes include the peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991). One probe of the present invention detects a mutation of a codon corresponding to one amino acid mutation at a specific site. The mutation type should have a difference in hybridization strength so as not to hybridize to other nucleotide sequences other than the mutation, It should be strict enough to hybridize only to mutations. The markers according to the present invention can be provided in a form preliminarily bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and conditions of 25-30 [deg.] C may be suitable for, but are not limited to, hybridization for detection of mutations.

The process of immobilizing a polynucleotide used as a probe according to the present invention on a substrate can also be easily manufactured using such a conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

In another aspect, the invention is directed to a method for detecting liver disease diagnostic markers according to the present disclosure to provide information necessary for the diagnosis or prognosis of liver disease, including hepatocellular carcinoma. Detection of the marker can be performed by confirming the presence of nucleic acid or protein.

In one embodiment, the method comprises the steps of: i) obtaining a nucleic acid sample from a subject in need of a liver disease, particularly a hepatocellular carcinoma test, in particular a patient infected with HBV; ii) the mutation (C-P5H / L / T) of the 5th amino acid of the core protein C region of HBV in the obtained nucleic acid sample, the mutation of the 83rd amino acid (C-E83D) / L), a mutation (C-L100I) of the 100th amino acid, a mutation (C-Q182K / *; C-Q184K / *) of the 182nd or 184th amino acid and / *), ≪ / RTI > And iii) at least one codon mutation corresponding to C-P5H / L / T, C-E83D, C-I97F / L, C-L100I, C-Q182K / * or preC- , Which is associated with the diagnosis or prognosis of liver disease, for example, hepatocellular carcinoma.

The amino acid residue number of the core protein preC and C region of HBV depends on the order of residues of HBV genotype A to H type, and the specific sequence and residue variation of preC and C region according to the type are as mentioned above. The specific amino acid residues of the core protein preC region and the C region of the HBV are as described above.

Detection of a marker according to the present invention may be performed by detecting the presence or absence of a nucleic acid or protein, particularly a nucleic acid of a certain base sequence.

 The step of obtaining the nucleic acid from the specimen of step (i) above may be carried out by a conventional DNA separation method. The nucleotide sequence determination in step (ii) may be performed by sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH) , PCR extension analysis, or TaqMan technique. If the method involves amplification of the target nucleic acid region, it can be obtained by PCR amplification and purification. Other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

The specimen used in the method of the present invention is as described above.

In the liver disease diagnosis marker of the present invention, a plurality of markers may be used in combination. Detection of the presence / absence of the marker in the method of the present invention can be determined at the level of protein and / or nucleic acid, and such is the same as mentioned above.

The method for detecting a marker for diagnosing liver cancer according to the present invention can discriminate the nucleotide sequence of the mutation site to predict the susceptibility of the liver cancer and provide information necessary for diagnosis of liver cancer.

Further, the method of the present invention may further use clinical information other than the marker analysis of the patient, in addition to the marker analysis result, in order to provide information on the diagnosis or prognosis as described above. Clinical information other than this marker analysis includes, for example, age, sex, weight, dietary habit, body mass, ultrasonography, CT, magnetic resonance imaging (MRI), angiography, endoscopic retrograde pancreatobiliary , Ultrasound endoscopy, tumor markers, or laparoscopy.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

The present invention may be practiced using conventional techniques that are within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, immunology, and the like. A more detailed description of common techniques can be found in the following books and literature. For general methods of molecular biology and biochemistry, see Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al., Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds., 1984); Culture of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., Eds.); And Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Immunochemical Methods In Cell & Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Protein Methods (Bollag et al., John Wiley & Sons 1996); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); And Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). For a general description of cell culture and media, see Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); And Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251). The reagents, cloning vectors and kits described herein can be purchased from companies such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

Example  One

Specimen

Serum samples were collected from 70 patients with chronic hepatitis B who visited Cheju National University Hospital in 2003 ~ 2009, Seoul Veterans Hospital in 2004 ~ 2006 or Seoul National University Hospital in 2005. Of the 35 serum samples, HBeAg-positive and 35 were HBeAg-negative. Clinical diagnoses were chronic hepatitis (CH) (n = 27), liver cirrhosis (CH) ) And hepatocellular carcinoma (HCC) (n = 35).

Patients with acute hepatitis B, hepatitis C or D virus infection, antiviral therapy, immunosuppressive therapy and severe alcohol abuse were excluded. HBsAg, anti-HBs, HBeAg and anti-HBe were analyzed using a commercially available enzyme immunoassay kit (Abbott Lab. Wiesbaden, Germany) according to the manufacturer's brochure. HBV DNA was qualitatively determined using a hybrid capture HBV DNA assay kit (Diagene, Gaithersburg, Md., USA) according to the manufacturer's instructions.

This study was approved by the institutional review board of Seoul National University Hospital (IRB No. C-1110-106-382). The clinical characteristics of the patients who participated in this study are as follows.

HBV DNA  Amplification and sequencing

In this example, virion DNA was isolated from the patient's serum and PCR was performed to generate 2698 nt of 1017 bp amplicon from 1682 nt of the HBV genome. For isolation of virion, 200 μg of serum was added to 600 μl of TES solution (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.5% SDS and proteinase 50 μg) and incubated at 65 ° C for 3 hours. DNA was then isolated using a mixture of phenol / chloroform / isoamylalcohol (50: 49: 1) and the DNA was precipitated with isopropyl alcohol. The DNA pellet was dissolved in 20 μl of TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 2 μl of the dissolved DNA was used for PCR.

 To analyze the frequency and variation patterns of deletion and insertion in the entire preC / C region, nested PCR was used. First, PCR was performed using a sense primer CoreF1 (5'-AAC GAC CGA CCT TGA GGC ATA CTT-3 ') and an antisense primer CoreR1 (5'-ATT TGG TAA GGT TAG GAT AGA A-3' And as a result, the HBV dielectric was amplified from 1682 nt to 2698 nt to obtain a 1017 bp product.

Secondary PCR was performed using the sense primer CoreF2 (5'-GAG TTG GGG GAG ATT AGG TTA-3 ') and the antisense primer CoreR2 (5'-CAC TCA GGA TTA AAG ACA G-3' A 822 bp product of 2555 nt at 1734 nt of the HBV genome was generated.

PCR was initiated in a 50 μl PCR mixture containing 1.5 mM MgCl 2, 200 μM dNTP, and 2.6 U of Expand High Fidelity Taq polymerase. Both experimental conditions were 95 ° C for 10 min; 20 cycles consisting of 95 캜 (45 sec), 52 캜 (45 sec) and 72 캜 (90 sec); Lt; RTI ID = 0.0 > 72 C < / RTI > for 5 minutes. 1 to 5 μl of the product was used, and the second PCR was also performed once.

PCR products were analyzed by electrophoresis on 2.5% agarose gel, stained with ethidium bromide and visualized with UV. The purified PCR products were sequenced using CoreF2 and CoreR2 primers used for the second PCR using BigDye Terminator Cycle Sequencing Ready reaction V.2 kit and fluorescent 373A DNA sequencer (Applied Biosystems, Foster City, CA, USA) Respectively. When sequences are mixed, the dominant one is adopted.

HBV  Genotype determination

For genotyping, a phylogenetic analysis based on the entire sequence of the entire preC / C region (639 bp) was performed for all 70 HBV strains. Phylogenetic analysis was determined using neighbor-joining of MEGA version 4.1.

Statistical analysis

Results were expressed as a percentage, mean ± SD or median (range). The differences between the categorical variables were analyzed using Fisher's exact probability test or Chi-square test. Student t test was used when the data showed a normal distribution for the continuous variable, and Mann-Whitney U test was used when the data did not show a normal distribution. A p-value less than 0.05 is considered statistically significant.

Experiment result

Genotypic crystal distribution

In a phylogenetic analysis based on all 639 bp sequences in the preC / C region, all 70 HBV strains derived from Korean patients, regardless of clinical status and HBeAg serum status, appeared to belong to genotypic C2 (results not shown).

HBV preC / Distribution of C site mutations

Variations were found in a total of 83 codons among 212 codons (29 codons in preC, 183 codons in C) from 60 patients (85.7%) of 70 patients (see Figure 1, Table 1). In general, patients with mutations in the free C / C region (60 patients) were significantly older (51.9 vs. 36.9, P <0.001) than patients without mutations (10 patients). No other clinical factors leading to significant differences were found between the two groups (see Table 1).

[Table 1] Comparison of clinical characteristics of patients according to wild type and mutation

No deletions were found in the preC / C region of 70 patients except for displacement (data not shown). There was a non-random distribution in the preC / C site mutations. The mean value of all variants in the entire preC / C region from 70 HBV strains was 2% (4.2 of the 212 amino acids). The rate of variability in the MHC class I restriction sites (named M1RR) (2.2%) or the class II restriction sites (named M2RR) (2.3% ) (P <0.001), respectively. This was more pronounced when considering the variability (4.1%) on the hotspot, the amino acid residues 81-105 in the MHC class II restriction site (Table 2).

[Table 2] Comparison of variation rates among four regions in HBcAg

간질환 진행도와 관련되고 HBeAg 생산의 억제에 이르게 하는 28번째 코돈 내 변이(트립토판에서 종결코돈, preC-W28*로 표기)는 preC 부위에서 가장 빈번하게 발견된다(17 환자, 24.3%). C 부위에서, 101번째 코돈 내 변이(루신에서 트립토판 또는 세린, C-L101W/S로 표기)는 가장 빈번하게 발견되었다 (22환자, 31.4%)(도 1).

The 28th codon mutation (termination codon in tryptophan, preC-W28 *) associated with liver disease progression and leading to inhibition of HBeAg production is most frequently found in the preC region (17 patients, 24.3%). In the C region, the 101st codon mutation (expressed as tryptophan or serine, C-L101W / S in leucine) was found most frequently (22 patients, 31.4%) (Figure 1).

HCC  Patients and Control Patients ( LC + CH ) Frequency of variation between

The overall frequency of preC / C mutations in HCC patients (2.2%) was higher than in control patients (liver cirrhosis LC + chronic hepatitis CH) (1.8%) (P = 0.061). There was no difference in the frequency of mutations in the NRR between the two clinical groups (0.7% in HCC, 0.8% in the control). However, the rate of change in M2RR (2.7% vs. 1.9%, P = 0.024), not in M1RR (2.5% vs. 1.9%, p = 0.248), was significantly higher in HCC patients than in control patients. Furthermore, the difference at the amino acid residues 81-105 was more pronounced (5.6% vs. 2.6%, P = 0.002) (Table 3). This indicates that the frequency of mutations in M2RR is increased due to the immune evasion phenomenon as the lesion progresses after the virus infection.

[Table 3] Comparison of Mutation Frequency of HCC Patients and Control Patients (LC + CH) in 4 Areas in HBcAg

Two different HBeAg  Having a serum state Between patient  Frequency of variation

Overall, the overall frequency of preC / C mutations was significantly higher in the HBeAg negative group (2.5%) than in the HBeAg positive group (1.5%) (P <0.001). However, some differences were found for each region in HBcAg. The difference between the two groups was not statistically significant in NRR variability (0.9% vs. 0.6%, P = 0.144), but M1RR (2.6% vs. 1.7%, P = 0.094) and M2RR (3.0% versus 1.7%, P <0.001), especially the percentages of mutations in the amino acid residues 81-105 (5.0%) were significantly higher in the negative patients than in the HBeAg positive patients, respectively. M2RR (3.0%) was more sensitive to mutations induced by HBeAg seroconversion than M1RR (2.6%) (Table 4). This indicates that HBeAg negative patients, especially with respect to M2RR, exhibit immune avoidance against cytotoxic T cells.

[Table 4] Comparison of mutation frequencies between two HBeAg serotypes in four regions of HBcAg

HCC Related to preC Identification of mutation patterns in the / C region

(C-P5H / L / T, C-E83D, C-I97F / L, C-L100I and C-Q182K / -W28 *) were found to be associated with HCC patients by comparison with patients at different stages of the disease, such as LC and CH, respectively. In general, the variation at the 5 sites at the C site was found to be significantly higher in HCC patients than in the control patients at other stages of the disease. In addition, preC-W28 * prodominance in HCC patients was close to statistically significant (P = 0.093) (Fig. 2a).

Notably, four of the five HCC-associated C region mutations (C-P5H / L / T, C-E83D, C-I97F / L, C-L100I) were located in MHC class II restricted T cell epitopes. Of the six HCC-associated preC / C mutations, C-I97F / L, which is known to lead to defects in HBcAg assembly, was found most frequently in HCC patients (13 patients). A comparison of clinical data between patients with and without C-I97F / L showed that this mutation was associated with older (57.4 vs. 47.3, P = 0.01) and lower levels of HBV DNA (29300 vs. 5534304 P = 0.084) (Table 5)

[Table 5] Comparison of clinical features between wild type and patients with I97F / L mutation

HBeAg  Affect the serum state preC Identification of mutation patterns in the / C region

(C-D32N / H, C-E43K, C-P50A / H / Y, C-A131G / N / P and C-S181H / P) (PreC-W28 *, preC-G29D) was found to affect HBeAg serum status (Figure 2b). (C-D32N / H, C-E43K) and five other mutations (preC-W28 *, C-G29D, C-P50A / H / Y, C-A131G / N / S181H / P) were found to be associated with HBeAg positive and HBeAg negative serum, respectively. These markers were all statistically significant or close to significant levels of significance (C-G29D, C-P50A / H / Y, C-A131G / N / P, C-S181H / P) , C-D32N / H). Interestingly, two spatially related HBeAg mutations (C-P50A / H / Y, C-A131G / N / P) were located in the M2RR at the C site (FIG.

HBV genotyping and sequencing of the preC / C mutation in a total of 70 Korean patients, including 35 with HCC, confirmed that all patients had a genotype C infection. Variations of the six types (preC-W28 *, C-P5H / L / T, C-E83D, C-I97F / L, C-L100I, C-Q182K / *) in the preC / Particularly in the MHC class II restricted region.

This indicates that mutations in the C region and preC region of the HBV core protein identified herein can be useful for, among other things, chronic hepatitis B infection in genotype C, progression to HCC in cirrhotic patients, and HCC diagnosis.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> SNU R & DB Foundation <120> Markers for diagnosing liver disease and its use <130> P0002 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 29 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE <222> (1) (29) <223> HBV genotype C preC region <400> 1 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr   1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly              20 25 <210> 2 <211> 183 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE &Lt; 222 > (1) .. (183) <223> HBV genotype C region <400> 2 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu   1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp              20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys          35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu      50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala  65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys                  85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg             100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr         115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro     130 135 140 Glu Thr Th Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Arg Arg Arg Arg Ser Ser Gln Ser Pro Arg Arg Arg Arg Ser                 165 170 175 Gln Ser Arg Glu Ser Gln Cys             180 <210> 3 <211> 87 <212> DNA <213> Hepatitis B virus <220> <221> gene <222> (1). (87) <223> HBV genotype C preC region <400> 3 atgcaacttt ttcacctctg cctaatcatc tcatgttcat gtcctactgt tcaagcctcc 60 aagctgtgcc ttgggtggct ttggggc 87 <210> 4 <211> 552 <212> DNA <213> Hepatitis B virus <220> <221> gene &Lt; 222 > (1) .. (552) <223> HBV genotype C region <400> 4 atggacattg acccgtataa agaatttgga gcttctgtgg agttactctc ttttttgcct 60 tctgacttct ttccttctat tcgagatctc ctcgacaccg cctctgctct gtatcgggag 120 gccttagagt ctccggaaca ttgttcacct caccatacag cactcaggca agctattctg 180 tgttggggtg agttgatgaa tctggccacc tgggtgggaa gtaatttgga agacccagca 240 tccagggaat tagtagtcag ctatgtcaac gttaatatgg gcctaaaaat cagacaacta 300 ttgtggtttc acatttcctg tcttactttt ggaagagaaa ctgttcttga gtatttggtg 360 tcttttggag tgtggattcg cactcctccc gcttacagac caccaaatgc ccctatctta 420 tcaacacttc cggaaactac tgttgttaga cgccgaggca ggtcccctag aagaagaact 480 ccctcgcctc gcagacgaag gtctcaatca ccgcgtcgca gaagatctca atctcgggaa 540 tctcaatgtt ag 552 <210> 5 <211> 29 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE <222> (1) (29) <223> HBV genotypeA preC region <400> 5 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Thr Cys Pro Thr   1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly              20 25 <210> 6 <211> 185 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE <222> (1). (185) <223> HBV genotypeA C region <400> 6 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu   1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp              20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys          35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Val Glu      50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala  65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys                  85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg             100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr         115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro     130 135 140 Glu Thr Th Val Val Arg Arg Arg Asp Arg Gly Arg Ser Ser Arg Arg 145 150 155 160 Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg                 165 170 175 Arg Ser Gln Ser Arg Glu Ser Gln Cys             180 185 <210> 7 <211> 87 <212> DNA <213> Hepatitis B virus <220> <221> gene <222> (1). (87) <223> HBV genotypeA preC region <400> 7 atgcaacttt ttcacctctg cctaatcatc tcttgtacat gtcccactgt tcaagcctcc 60 aagctgtgcc ttgggtggct ttggggc 87 <210> 8 <211> 558 <212> DNA <213> Hepatitis B virus <220> <221> gene <222> (1) .. (558) <223> HBV genotypeA C region <400> 8 atggacattg acccttataa agaatttgga gctactgtgg agttactctc gtttttgcct 60 tctgacttct ttccttccgt cagagatctc ctagacaccg cctcagctct gtatcgagaa 120 gccttagagt ctcctgagca ttgctcacct caccatactg cactcaggca agccattctc 180 tgctgggtgg aattgatgac tctagctacc tgggtgggta ataatttgga agatccagca 240 tccagggatc tagtagtcaa ttatgttaat actaacatgg gtttaaagat caggcaacta 300 ttgtggtttc atatatcttg ccttactttt ggaagagaga ctgtgcttga atatttggtc 360 tctttcggag tgtggattcg cactcctcca gcctatagac caccaaatgc ccctatctta 420 tcaacacttc cggaaactac tgttgttaga cgacgggacc gaggcaggtc ccctagaaga 480 agaactccct cgcctcgcag acgcagatct caatcgccgc gtcgcagaag atctcaatct 540 cgggaatctc aatgttag 558 <210> 9 <211> 29 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE <222> (1) (29) <223> HBV genotypeB preC region <400> 9 Met Gln Leu Phe His Leu Cys Leu Val Ile Ser Cys Ser Cys Pro Thr   1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly              20 25 <210> 10 <211> 183 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE &Lt; 222 > (1) .. (183) <223> HBV genotype B C region <400> 10 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu   1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp              20 25 30 Thr Val Ser Ala Leu Tyr Arg Glu Ala Leu Lys Ser Pro Glu His Cys          35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu      50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala  65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys                  85 90 95 Ile Arg Gln Leu Trp Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg             100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr         115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro     130 135 140 Glu Thr Th Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Arg Arg Arg Arg Ser Ser Gln Ser Pro Arg Arg Arg Arg Ser                 165 170 175 Gln Ser Arg Glu Ser Gln Cys             180 <210> 11 <211> 87 <212> DNA <213> Hepatitis B virus <220> <221> gene <222> (1). (87) <223> HBV genotypeB preC region <400> 11 atgcaacttt ttcacctctg cctagtcatc tcttgttcat gtcctactgt tcaagcctcc 60 aagctgtgcc ttgggtggct ttggggc 87 <210> 12 <211> 552 <212> DNA <213> Hepatitis B virus <220> <221> gene &Lt; 222 > (1) .. (552) <223> HBV genotype B C region <400> 12 atggacattg acccctataa agaatttgga gctactgtgg agttactctc ttttttgcct 60 tctgacttct ttccgtcggt gcgggacctc ctagataccg tctctgctct gtatcgggaa 120 gccttaaaat ctcctgagca ttgctcacct caccacacag cactcaggca agctattctg 180 tgctgggggg aattaatgac tctagctacc tgggtgggta ataatttgga agatccagca 240 tcccgggatc tagtagtcaa ttatgttaac actaacatgg gcctaaagat caggcaacta 300 tggtggtttc acatttcctg tcttactttt ggaagagaaa ctgttctgga atatttggta 360 tcttttggag tgtggattcg cactcctcct gcctacagac caccaaatgc ccctatctta 420 tcaacacttc cggaaactac tgttgttaga cgacgaggca ggtcccctag aagaagaact 480 ccctcgcctc gcagacgaag gtctcaatca ccgcgtcgca gaagatctca atctcgggaa 540 tcccaatgtt ag 552 <210> 13 <211> 29 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE <222> (1) (29) <223> HBV genotype D preC region <400> 13 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr   1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly              20 25 <210> 14 <211> 183 <212> PRT <213> Hepatitis B virus <220> <221> PEPTIDE &Lt; 222 > (1) .. (183) <223> HBV genotypeD C region <400> 14 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu   1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp              20 25 30 Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys          35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu      50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala  65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys                  85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg             100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr         115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro     130 135 140 Glu Thr Th Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Arg Arg Arg Arg Ser Ser Gln Ser Pro Arg Arg Arg Arg Ser                 165 170 175 Gln Ser Arg Glu Ser Gln Cys             180 <210> 15 <211> 87 <212> DNA <213> Hepatitis B virus <220> <221> gene <222> (1). (87) <223> HBV genotype D preC region <400> 15 atgcaacttt ttcacctctg cctaatcatc tcttgttcat gtcctactgt tcaagcctcc 60 aagctgtgcc ttgggtggct ttggggc 87 <210> 16 <211> 552 <212> DNA <213> Hepatitis B virus <220> <221> gene &Lt; 222 > (1) .. (552) <223> HBV genotypeD C region <400> 16 atggacattg acccttataa agaatttgga gctactgtgg agttactctc atttttgcct 60 tctgactttt ttccttcggt acgagatctt ctagataccg cctcagctct gtatcgggat 120 gccttagagt ctcctgagca ttgttcacct caccatactg cactcaggca agcaattctt 180 tgctgggggg aactaatgac tctagctacc tgggtgggtg ttaatttgga agatccagca 240 tctagggacc tagtagtcag ttatgtcaac actaatatgg gcctaaagtt caggcaacta 300 ttgtggtttc acatttcttg tctcactttt ggaagagaaa cagtcataga gtatttggtg 360 tctttcggag tgtggattcg cactcctcca gcttatagac caccaaatgc ccctatctta 420 tcaacacttc cggaaactac tgttgttaga cgacgaggca ggtcccctag aagaagaact 480 ccctcgcctc gcagacgaag atctcaatcg ccgcgtcgca gaagatctca atctcgggaa 540 tctcaatgtt ag 552 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Nested PCR 1st set: sense primer CoreF1 <400> 17 aacgaccgac cttgaggcat actt 24 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Nested PCR 1st set: antisense primer CoreR1 <400> 18 atttggtaag gttaggatag aa 22 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Nested PCR 2nd set: sense primer CoreF2 <400> 19 gagttggggg aggagattag gtta 24 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nested PCR 2nd set: antisense primer CoreR2 <400> 20 cactcaggat taaagacag 19

Claims (16)

At least one of histidine or leucine (C-P5H / L) in proline of the 5th amino acid of the core protein C region of HBV; Mutation of the 83rd amino acid from glutamic acid to aspartic acid (C-E83D); Mutation of the 97th amino acid from isoleucine to phenylalanine (C-I97F); Mutation of the 100th amino acid isosuric acid (C-L100I) in leucine; And a probe capable of detecting a polynucleotide comprising a site at which the 182nd or 184th amino acid encodes a codon mutation corresponding to at least one mutation in lysine or a stop codon (C-Q182K / *) in glutamine or a probe capable of amplifying the polynucleotide Or a probe and a primer,
The probes and primers may be used alone or in combination,
Wherein each of the amino acid residue numbers corresponds to a corresponding residue sequence of the C region of any one of HBV genotype A to H type.
delete The composition according to claim 1, wherein the liver disease is caused by A or C type HBV infection.
The composition for diagnosing liver diseases according to claim 1, wherein the core protein C region of the HBV has an amino acid sequence represented by SEQ ID NO: 2, 6, 10 or 14.
delete delete delete delete The composition for diagnosing liver disease according to claim 1, wherein the primer is a primer capable of amplifying the mutation site alone or two or more mutation sites.
10. The composition for diagnosing liver disease according to claim 9, wherein the primer is a pair of primers of SEQ ID NOs: 17 and 18, or a pair of primers of SEQ ID NOs: 19 and 20.
delete A kit for the diagnosis of liver disease comprising the composition according to any one of claims 1, 3, 4, 9 or 10.
13. The kit of claim 12, wherein the kit is for microarray analysis.
i) providing a nucleic acid sample from a patient infected with HBV;
ii) determining a nucleotide sequence of a site including codons corresponding to amino acids at positions 5, 83, 97, 100, and 182 or 184 of the core protein C region of HBV in the provided nucleic acid sample; And
(iii) a mutation in which the fifth amino acid of the core protein C region of the HBV is C-P5H or C-P5L, the mutation of the 83rd amino acid (C-E83D), the 97th amino acid When a codon mutation corresponding to the mutation (C-I97F) of the 100th amino acid (C-L100I) and the mutation of the 182nd or 184th amino acid (at least one of C-Q182 or 184K / Wherein the respective amino acid residue numbers are information necessary for the diagnosis or prognosis of a liver disease according to a corresponding residue sequence of a C region of any one of HBV genotype A to H type Way.
delete The method according to claim 14, wherein the nucleotide sequence analysis is performed by sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH) ), PCR extension assay, or TaqMan technique. &Lt; / RTI &gt;

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