KR100803258B1 - - Polynucleotides comprising single nucleotide polymorphism microarrays and diagnostic kits comprising the same and methods for diagnosing antibody-nonresponse to hepatitis B vaccine - Google Patents

Abstract

A polynucleotide comprising a single nucleotide polymorphism is provided to be usefully and effectively used for diagnosing the antibody-nonresponse to hepatitis B vaccine. A probe for diagnosing antibody-nonresponse to hepatitis B vaccine consists of at least one polynucleotide selected from the group consisting of a polynucleotide(b) consisting of more than 10 consecutive DNA sequences SEQ ID : NO. 2 including a 218th base(a polymorphic portion) of G; polynucleotide(b) consisting of more than 10 consecutive DNA sequences including a 218th base(a polymorphic portion) of G; a polynucleotide(c) consisting of more than 10 consecutive DNA sequences SEQ ID : NO. 3 including a 311st base(a polymorphic portion) of C; a polynucleotide(f) consisting of more than 10 consecutive DNA sequences SEQ ID : NO. 6 including a 301st base(a polymorphic portion) of C; and a polynucleotide(g) consisting of more than 10 consecutive DNA sequences SEQ ID : NO. 7 including a 294th base(a polymorphic portion) of A or a complementary nucleotide thereof. A microarray comprises the probe for diagnosing antibody-nonresponse to hepatitis B vaccine. A method for diagnosing the antibody-nonresponse to hepatitis B vaccine comprises the steps of: (a) obtaining a nucleic acid sample from a test body; and (b) determining the nucleotide sequence of at least one of the polymorphic portion among the polynucleotide of SEQ ID : NOs. 2, 3, 6 and 7 or the complementary nucleotide thereof.

Description

Polynucleotides comprising single nucleotide polymorphism, microarrays and diagnostic kits comprising the same, and methods for polynucleotides containing monobasic polymorphs, microarrays and diagnostic kits comprising the same, and hepatitis V vaccines using the same diagnosing antibody-nonresponse to hepatitis B vaccine}

1 is a diagram showing a haploid shape of an associative non-equilibrium block;

Figure 2 shows a haplotype formed in comparison to genotyping results from associative non equilibrium blocks.

The present invention relates to polynucleotides or their complementary nucleotides comprising a monobasic polymorphism associated with an antibody-no response to a hepatitis B vaccine; Amplification primers for antibody-non-response diagnosis against hepatitis B vaccine consisting of the polynucleotide or its complementary nucleotides or antibody-non-response diagnostic probes for hepatitis B vaccine; A microarray comprising the probe; And an antibody-free response diagnostic method for hepatitis B vaccine using the polynucleotide or its complementary nucleotides.

The human genome consists of 22 pairs of homologous chromosomes and two sex chromosomes. But every human being has a different appearance and character. This is because, although individuals have the same number of chromosomes, the phenotypes of the genes expressed from the chromosomes are slightly different. The diversity of these gene expressions in individuals is due to the genetic diversity of the genome carrying the gene. This genetic diversity has been studied a lot until recently, the structure of the human genome has recently been revealed.

Genetic diversity in the genome is known as transposable elements, short tandem repeats (STRs), variable number tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs). Dual SNPs are monobasic and appear at a frequency of 2-3 of about 1000 nucleotides on the human genome and take the form of single nucleotide variations between individuals of the same species. The genetic meaning of these monobasic polymorphisms makes a big difference in each individual depending on its location. For example, when a monobasic polymorph is present at a position that encodes a protein, it may affect the structure of the protein and thus alter protein function, which may also be associated with disease. Representative examples are monobasic polymorphisms in the Apoie (APOE) and Braca (BRCA) genes. Apoy's monobasic polymorphisms are known to increase the risk of developing senile dementia and reduce the responsiveness to the drug, tarcrine. Braka is known to be associated with breast cancer.

The association between these genes and the disease is characterized by the location of the genes within the chromosome of monobasic polymorphism. For example, when present in exons can alter the structure and function of the protein and when present on other genes that do not encode the protein, i.e. when present in a promoter or intron, expression of the protein for each By varying levels, the overall activity of the protein can be reduced, and the protein can be abnormally expressed through alterative splicing.

Conventional techniques for the discovery of such monobasic polymorphisms include Restriction Fragment Length Polymorphism (RFLP) using restriction enzymes, TaqMan ^™ probe method using Allele-specific hybridization, and melting temperature ( Dynamic Allele-Specific Hybridization (DASH) using melting temperature (Tm), Pyrosequencing using polymerisation, and direct sequencing. Recently, microarray technology has integrated a single substrate on a small substrate using the principle of Allele-Specific Extension, and then hybridized with the target DNAs to find a single base polymorphism. have.

Hepatitis B is a viral disease caused by hepatitis B virus (HBV). In infants under 1 year of age, 90% of hepatitis B patients are chronic carriers. It is reported that 40% die from cirrhosis and liver cancer after 30 to 40 years. In other words, liver-related diseases caused by hepatitis B and liver cancer are closely related.

In general, the presence or absence of a vaccine responder can be diagnosed by using hepatitis B surface antigen (HBsAg) test and Anti-HB (antibody against hepatitis B surface antigen) test after the third vaccination. If you are not vaccinated after vaccination, you have a high chance of developing liver disease or liver cancer. However, early diagnosis of responders to vaccines by molecular biological methods has not been achieved. Therefore, if a single nucleotide polymorphism is identified that may be specific to an antibody-responsive patient to the hepatitis B vaccine, the antibody-response to the hepatitis B vaccine will be diagnosed. For this reason, there is a need in the art to find new monobasic polymorphic sites associated with antibody-no response to hepatitis B vaccines.

The present inventors conducted a study to find a single base polymorphism that is specific to the antibody-nonresponse to the hepatitis B vaccine. As a result, the inventors of the tumor necrosis factor receptor-associated factor 1 (TRAF 1) gene for the hepatitis B vaccine The discovery of a monobasic polymorphic site specific to antibody-nonresponsive patients has led to the completion of the present invention.

Accordingly, it is an object of the present invention to provide a polynucleotide or its complementary polynucleotide comprising a monobasic polymorphism associated with an antibody-no response to a hepatitis B vaccine.

In addition, an object of the present invention is to provide an amplification primer for the antibody-response diagnosis for hepatitis B vaccine consisting of the polynucleotide or its complementary nucleotides or an antibody-response diagnostic probe for hepatitis B vaccine. do.

Another object of the present invention is to provide a microarray including the probe.

It is also an object of the present invention to provide an antibody-non-response diagnostic method for a hepatitis B vaccine using the polynucleotide or its complementary nucleotides.

According to one aspect of the invention, in the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, the polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 218th base (polymorphic site) is G, and comprising said 218th base ( b); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, the polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 311 th base (polymorphic site) is C and comprising the 311 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, a polynucleotide (f) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) C and comprising the 301th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, wherein the 294th base (polymorphic site) is A and a polynucleotide (g) consisting of 10 or more consecutive DNA sequences comprising the 294th base. Polynucleotides selected above or complementary nucleotides thereof are provided.

In addition, according to another aspect of the present invention, there is provided a primer for amplification for antibody-response diagnosis against hepatitis B vaccine, consisting of the polynucleotide or its complementary polynucleotide.

In addition, according to another aspect of the present invention, there is provided an antibody-non-response diagnostic probe for a hepatitis B vaccine consisting of the polynucleotide or its complementary polynucleotide, and a microarray comprising the same.

In addition, according to another aspect of the present invention, there is provided a kit for antibody-response diagnosis against hepatitis B vaccine, comprising the polynucleotide or its complementary polynucleotide.

In addition, according to another aspect of the present invention, there is provided an antibody-response diagnostic method for a hepatitis B vaccine using the polynucleotide or its complementary polynucleotide.

Hereinafter, the present invention will be described in detail.

As used herein, “antibody-no response to hepatitis B vaccine” refers to hepatitis B surface antigen (HBsAg) test, Anti-HB (antibody to hepatitis B surface antigen), and after hepatitis B vaccination; In the titer (Radioimmunoassay units) test, the titer is less than 100 U / ml with no antigen response and no response to hepatitis surface antigen. In addition, as a relative term for the term, antibody-normal response (ie, normal) to hepatitis B vaccine refers to the case where the titer is 1000 U / ml or more.

The present invention provides polynucleotides or complementary nucleotides thereof selected from the group consisting of polynucleotides (b), (c), (f) and (g) associated with antibody-no response to a hepatitis B vaccine. .

The DNA sequences of SEQ ID NOs: 2, 3, 6, and 7 are single nucleotide polymorphisms showing statistically significant difference (significance level p value <0.05) in the antibody-non-responder group and the normal group against the hepatitis B vaccine. They are present in the 5 'and 3' UTRs of the tumor necrosis factor receptor-associated factor 1 gene. TRAF1 is TNFR2, CD27, CD30, 4-1BB, OX40, HVEM, TRN (TNFR-associated death domain protein), TANK (TRAF-associated and NF-κB activator), TRIP (TRAF-interacting protein), NIK (NF- κB-inducing kinase, RIP (receptor-interacting protein), RIP2 (Cardiak), A20 (the NF-κB inhibitory protein), cIAP1 (the apoptosis-suppressors inhibitor of apoptosis 1), cIAP2, FLIP (FADD-like interleukin- It is a pivotal signal transduction agent that binds to a signal transduction agent such as 1β converting enzyme (FLICE) -like inhibitory protein), and it is surprising that the monobasic polymorphism is associated with antibody-response to hepatitis B vaccine. will be.

The present invention includes a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (b), (c), (f) and (g) or complementary nucleotides thereof, and the polynucleotide or complementary thereof The red nucleotide is 10 to 100 nucleotides in length, preferably 20 to 60 nucleotides, more preferably 40 to 60 nucleotides.

The polynucleotide according to the invention is at least one polynucleotide selected from the group consisting of (b), (c), (f) and (g) is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a monobasic polymorphism in a nucleotide sequence. A polymorphic site refers to a site where a monobasic polymorphism occurs in the polymorphic sequence. The polynucleotide can be DNA or RNA.

In the present invention, at least one polynucleotide selected from the group consisting of the polynucleotides (b), (c), (f) and (g) is involved in antibody-no response to the hepatitis B vaccine. This was confirmed by analyzing the genotypes of alleles in each monobasic polymorphism in genomic DNA obtained from blood samples derived from two groups of hepatitis antibodies. In the present invention, the normal and the patient group participated in each of 15 people, and after the third hepatitis vaccine, normal (high responder) with hepatitis B surface antigen (HBsAg) test and Anti-HB (antibody to hepatitis B surface antigen) ) And patient group (US / Low Responder), and each responder was used for data analysis along with epidemiological surveys such as age and Body Mass Index (BMI). The size of the cluster used for genotyping is shown in Table 1 below.

group Normal people Patient group total Survey 15 15 30

Tables 2 and 3 below show the association of the DNA sequences of SEQ ID NOS: 1-7 with the antibody-no response to the hepatitis B vaccine and the properties of the polymorphic sequences.

In Table 2a and Table 2b, what each column means is as follows.

SNPs represent bases observed at sites of monobasic polymorphism.

Sequence number is a sequence number which represents each monobasic polymorphism.

The analysis method used Infinium ^TM Assay method of Illumina.

The samples of the antibody-non-responders and normal responders to the hepatitis vaccine were subjected to serological methods after administration of the third vaccine after receiving consent from the volunteers, and the normal responder (control) selected a case where the antibody formation was 1000 U / ml or more In the case of no / low responder (case) genotype was analyzed using a total of 30 samples, each of 15 subjects less than 100 U / ml. The number of samples used here is divided into case and control. The allele frequency represents the frequency of alleles in case A2 (case A2) and con A2 (control A2), respectively.

The genotype frequency is expressed in terms of A1A1 (Major Homozygote), A1A2 (Heterozygote) and A2A2 (Minor Homozygote), and the number of people with genotypes A1A1, A1A2 and A2A2 in the case and control groups. Indicates.

The HWE state represents the state of Hardy-Weinberg Equilibrium, and con_HWE and cas_HWE represent the state of Hardy-Wineberg equilibrium in normal and diseased groups. Since it is determined to be Hardy-Weinberg Disequilibrium, and if the value is large, Hardy-Weinberg Equilibrium is determined. The analysis shows that the Hardy-Wineberg equilibrium value is the Hardy-Wineberg equilibrium because both the case and control values are above 0.05.

Table 3 shows the differences between the groups using the sum of the respective genotypes divided into genetically classifiable alleles and dominant, recessive, and co-dominant (co-dominant). Confirmed.

A dominant genotype was selected as a dominant genotype, and when the dominant allele is A and the recessive allele is a, the allele is the A [(( A × 2) + Aa) / (2 × N)] and a [((a × 2) + Aa) / (2 × N)], the dominant compares the sum of AA and Aa and aa Recessive was compared by the sum of AA versus Aa and aa. Codominant was analyzed by classifying each genotype of AA, Aa, aa.

The odds ratio represents the ratio of the probability of having a risk allele among the normal group to the probability of having a risk allele among the patient group. The odds ratio can be calculated as the ratio of odds between the case and the control. Oz in Case is [(frequency of A2A2 genotype × 2 + frequency of A1A2 genotype) / (number of samples in disease group × 2)] and oz for Control is [(frequency of A2A2 genotype × 2 + frequency of A1A2 genotype) / (normal group) Number of samples x2)]. For each oz obtained through the above equation, the odds ratio can be obtained by dividing the case with the control.

CI (Woolf 'Confidence interval) represents 95% confidence interval of the odds ratio, and each lower confidence interval is 100 (1-α)% CI = OR ^ 1- (Zα / √X ^ 2), upper confidence interval Is calculated as 100 (1-α)% CI = OR ^ 1 + (Zα / √X ^ 2)). In general, confidence intervals of 1 do not indicate a significant association with the disease.

The interpretation of the odds ratio is as follows. An odds ratio of 1 means no difference between the two groups. If the odds ratio is greater than 1 and no 1 is included in the 95% confidence interval, the disease appears at a higher frequency than the control. If the odds ratio is less than 1, it means that the comparative gene in the control group has a protective effect.

More accurate values can be confirmed by the chi-exact test and the p-value by chi-square. Chi-square is a method used to identify homogeneity, independence, and suitability between two groups, and the difference between expectations and observations can be observed. The p-value can be found from the chi-square variance table. In a scatter table, p-values vary depending on the value of degrees of freedom (df = n-1). In case of allele, dominant, recessive, two genotypes are compared, so n-1 = 2-1 = 1, so that p-value is obtained at 1 degree of freedom, In the case of co-dominant), since genotypes are AA, Aa, aa, and n-1 = 3-1 = 2, find and use the p-value at 2 degrees of freedom.

Fisher's exact test is a value used to test the statistical significance (p-value) of the chi-square value. In the present invention, when p-value ≦ 0.05, it was determined that the genotype of the disease group and the normal group was not the same.

The risk allele is A1 as the reference allele and A1 is the risk allele if the frequency of A1 in the disease group is greater than that of A1 in the normal group (ie, cas_A1> con_A1), and vice versa. In this case, A2 was used as a risk allele.

According to the detailed description of Table 3, the analysis of the genotype of the patient group and the normal group for each allele, dominant, recessive and co-dominant is as follows.

The allele can identify how risk alleles are associated with disease through the ratio of the frequency at which alleles appear between patients and normal subjects. SEQ ID NOS: 1, 4, and 5 have no significance because they contain 1 when they contain 1 in the 95% confidence interval. The remaining SEQ ID Nos. 2, 3, 6, and 7 are significant because they do not include 1 because they have confidence intervals 1.5705-13.8667 in the comparison between the groups, and have alleles of G, C, C, and A, respectively. Because the odds ratio has a high frequency of 4.6667 times, alleles of G, C, C, and A can be considered as risk alleles in each monobasic polymorph.

Chi-square was used to analyze whether the two alleles had statistically significant differences in the frequency of antibody-free and low-responder and high-responder groups for hepatitis vaccines. Here, the significance level (rejection region) was set to p_value <0.05, and the frequency of occurrence of the alleles of each monobasic polymorphism in the patient group and the normal group showed that the G allele of the risk allele of SEQ ID NO: 2, SEQ ID NO: The C allele, the C allele of SEQ ID NO: 6, and the A allele of SEQ ID NO: 7 were identified as having a significant p-value of 0.004486 in the Chi-Square test results. In addition, in order to test the statistical significance (p-value) of Chi-Square test, it was confirmed that Fisher's exact result shows that the p-value between the two groups has a very accurate Chi-Square test value. Here, chi-exact-p_value (chi-exact-p_value) is a value obtained from a chi-square distribution table having a degree of freedom (df) of 1. In the chi-square test, if the expected frequency is less than 5, the general chi-square test result may be inaccurate. Therefore, this variable is used to test statistical significance more accurately through Fisher's exact test.

The interpretation of the analysis results using the dominant trait is as follows.

SEQ ID NOS: 1, 4, and 5 have no significance because they contain 1 when they contain 1 in the 95% confidence interval. Remaining SEQ ID Nos. 2, 3, 6, and 7 have a confidence interval of 1.2678-118.3664, and thus they are significant because they do not contain 1, and SEQ ID No. 2 (G / G + G / A of dominant). ), 3 (C / C + C / T), 6 (C / C + C / G), and 7 (A / A + A / G) have a 12.25-fold difference in odds ratio between groups. It can be seen as a risk allele.

The significance level of the chi-square (rejection region) was confirmed under the same conditions as the allele, and the frequency in the patient group and the normal group showed G / G + G / A in SEQ ID NO: 2 In the Chi-Square test results of the C / C + C / T risk factor of 3, the C / C + C / G risk allele of 6 and the dominant trait of A / A + A / G No. 7, the p-value was 0.0132. The significance was confirmed. In addition, the results of Fisher's exact test to test the statistical significance (p-value) of the Chi-Square test showed that the p-value between the two groups had a very accurate Chi-Square test value of 0.0352.

Interpretation of the analysis results using recessive traits is as follows.

Since 1 contains 95% confidence interval, there is no significance because all of SEQ ID NOs 1 to 7 contain 1.

Interpretation of the analysis results using codominant traits is as follows.

SEQ ID NOS: 1, 4, and 5 are not significant because they contain 1 in the 95% confidence interval. Remaining G / G; G / A; A / A of SEQ ID NO: 2, C / C of SEQ ID NO: 3; C / T; C / C; C / C; C / G; G / G of SEQ ID NO: 6, sequence Significant for A / A; A / G; G / G in number 7. In the case of co-trait genes, the p-value was found to be significant as 0.0253 in Chi-Square test results using the risk alleles of SEQ ID NOs: 2, 3, 6, and 7, respectively. Also, to test the statistical significance (p-value) of Chi-Square test, Fisher's exact result shows that Chi-Square test value is very accurate with 0.028 p-value between two groups.

Marker Name Reference Sequence chromosome band location gene NM number Position in the gene Amino acid change Gene description CHAHBV001 rs2416806 chr9 9q33.2 120769846 TRAF1 NM_005658 flanking_5UTR radish In the process of signal transmission, it acts as an intermediate to deliver the signal received from the receptor to mediate signal transmission. CHAHBV002 rs3761847 chr9 9q33.2 120769793 TRAF1 NM_005658 flanking_5UTR radish CHAHBV003 rs3761846 chr9 9q33.2 120769151 TRAF1 NM_005658 flanking_5UTR radish CHAHBV004 rs7021880 chr9 9q33.2 120753444 TRAF1 NM_005658 intron radish CHAHBV005 rs2239657 chr9 9q33.2 120751074 TRAF1 NM_005658 coding radish CHAHBV006 rs2241003 chr9 9q33.2 120746331 TRAF1 NM_005658 3UTR radish CHAHBV007 rs9886724 chr9 9q33.2 120744573 TRAF1 NM_005658 3UTR radish

The meaning of each column in Table 4 is as follows. Columns such as chromosomes, bands, genes, descriptions, monobasic polymorphisms (SNPs), amino acid changes, etc. are annotations for each monobasic polymorphism.

The marker name is a number added for convenience to the single base polymorph found.

Rs stands for Reference Sequence (RS), which is the name given after the Human Genome Project to identify each single nucleotide polymorphism in the database of the National Institute of Biotechnology Information (NCBI).

In chromosome, the number of chromosomes in which the single nucleotide polymorphisms are located.

The position column indicates the number of bases in which the single nucleotide polymorphisms are located when the entire nucleotide base constituting the chromosome 6 is numbered.

The gene represents a gene in which the single nucleotide polymorphisms are located, and is located in a gene called TRAF1.

The location column in the gene indicates where the single base polymorphisms are located on the gene (TRAF1). Thus, it can be seen how each monobasic polymorphism works in gene expression and function.

A change in amino acid is a description of whether a gene is expressed by the single nucleotide polymorphisms to change the amino acids constituting the protein. Herein, all of the single nucleotide polymorphisms encode a protein. Since it does not exist in exon, it is irrelevant to changes in amino acids.

Gene description is a description of the biochemical and metabolic activity of the gene called TRAF1, indicating the main function of this gene. Based on this, you can find out how it affects the disease.

As shown in Tables 2-4, the polymorphic markers of SEQ ID NOs: 2, 3, 6, and 7 of the present invention are alleles (SEQ ID NO: 2: G, SEQ ID NO: 3: C, SEQ ID NO: 6: C). , And SEQ ID NO: 7: A), Dominant (SEQ ID NO: 2: G / G + G / A, SEQ ID NO: 3: C / C + C / T, SEQ ID NO: 6: C / C + C / G, And SEQ ID NO: A / A + A / G), and Codominant (SEQ ID NO: 2: G / G; G / A; A / A, SEQ ID 3: C / C; C / T; T / T, SEQ ID NO: 6 C / C; C / G; G / G, and SEQ ID NO: 7: A / A; A / G; G / G), antibody-no-responder to hepatitis vaccine between expectations and observations And the frequency of appearance in the normal responders was found to be related. That is, since the monobasic polymorphisms of SEQ ID NOs: 2, 3, 6, and 7 showed significant frequency of appearance in the antibody-non-responsive patient group and normal subjects against the hepatitis B vaccine, the monobasic polymorphism was It can be effectively used for diagnosis or treatment associated with antibody-no response to. Specifically, it can be used as a primer or probe for diagnosis of antibody-no response to hepatitis B vaccine.

The invention also provides for antibody-response diagnostics for hepatitis B vaccines that hybridize with at least 10 consecutive polynucleotides or their complementary nucleotides derived from the DNA sequences of SEQ ID NOs: 2, 3, 6, and 7, including polymorphic sites. Allele specific polynucleotides. The allele-specific polynucleotide means to hybridize specifically to each allele. That is, it means that the single base polymorphism can specifically hybridize to one of the alleles represented, wherein hybridization may be performed at 48 ° C. under RA1 solution (Illumina's InfiniumTM Assay kit).

The present invention also includes an allele specific primer for amplification for antibody-nonresponsive diagnosis for hepatitis B vaccine, consisting of polynucleotides or complementary polynucleotides thereof. “Primer” herein refers to the synthesis of template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates (ATP, GTP, CTP, TTP) and DNA polymerase) in a suitable buffer and at a suitable temperature. It refers to a single stranded oligonucleotide that can act as a starting point. The appropriate length of the primer may vary depending on the purpose of use, but is usually 10 to 100, preferably 10 to 80 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. Preferably, the primer is aligned with the single nucleotide polymorphism of the 3'-end of SEQ ID NO: 2, 3, 6, and 7. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification in allelic form of DNA where the primer exhibits complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. The product is amplified from two primers by amplification, and only the amplified product is specifically stained and visualized. This means that certain allelic forms exist. If the allele-specific polynucleotides used herein undergo different denaturation processes, they may be used by other methods as well as Infinium ^™ Assay.

The present invention is for diagnosing antibody-response against hepatitis B vaccine consisting of at least one polynucleotide selected from the group consisting of polynucleotides (b), (c), (f), and (g) or complementary polynucleotides thereof. Probes and antibody-response diagnostic microarrays for hepatitis B vaccines comprising the same. The present invention also provides an antibody against a hepatitis B vaccine comprising at least one polynucleotide selected from the group consisting of polynucleotides (b), (c), (f), and (g) or complementary polynucleotides thereof. Non-response diagnostic kits.

The present invention comprises the steps of obtaining a nucleic acid sample from a sample and determining the nucleotide sequence of one or more polymorphic sites of the polynucleotides of SEQ ID NOs: 2, 3, 6, and 7 or complementary nucleotides thereof, wherein the polymorphic sites are Anti-response diagnostic methods for hepatitis B vaccines, as defined).

Here, the step of determining the nucleotide sequence of the polymorphic site is a polynucleotide selected from the group consisting of polynucleotides (b), (c), (f), and (g) or a complementary nucleotides thereof. And hybridizing the nucleic acid sample and detecting the obtained hybridization result.

In another embodiment of the method of the present invention, when the nucleotide sequence of the polymorphic site is determined, the bases of the polymorphic sites of SEQ ID NO: 2, 3, 6, and 7 are G, C, C, and A (risk alleles, respectively). If one or more) is present, it may further comprise the step of determining that belonging to the antibody-non-responsive group to the hepatitis B vaccine. The more the nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to the risk group can be determined.

In another embodiment of the method of the present invention, the nucleotide sequence of the polymorphic site is determined, and as a result, the dominant genotypes of the polymorphic site of SEQ ID NO: 2, 3, 6, and 7 are G / G and G / A, respectively. , If at least one of C / C and C / T, C / C and C / G, and A / A and A / G (risk allele) is present, the antibody-responsive group to the hepatitis B vaccine The method may further include determining to belong. The more the nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to the risk group can be determined.

The present invention includes obtaining a nucleic acid sample from a sample and detecting the presence of a haplotype in a Tumor Necrosis factor receptor-Associated Factor 1 (TRAF 1) gene comprising the DNA sequences of SEQ ID NOs: 1-7. It includes a diagnostic method for the presence or absence of antibody-no response to the hepatitis B vaccine (Table 6, Figure 1-2).

In particular, the polymorphic site of the DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 7 (ie, 271 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1 218 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2; 311 base of the polynucleotide consisting of the DNA sequence of 3; 301 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, 501 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 5; If the haplotype analyzed from the 301th base of the constructed polynucleotide; and the 294th base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 7) is GCTGTAC, the case with Oz 4.67 in the results within the 95% confidence interval (1.57-13.87) The haplotype is B, since it has a haplotype of 4,67 times higher and also has a significant value of 0.0098 in the chi-square result. It can be preferably used for the diagnosis of non-presence of reaction-antibodies to the vaccine salt. Thus, according to another embodiment of the present invention, obtaining a nucleic acid sample from a sample; And a polymorphic site of the DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 7 (ie, 271 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1; 218 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2; SEQ ID NO: 3 311 base of the polynucleotide consisting of the DNA sequence of; 301 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, 501 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 5; The haplotype analyzed from the 301th base of the polynucleotide; and the 294th base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 7) is GCTGTAC, and belongs to the antibody-non-response group for hepatitis B vaccine. There is provided a diagnostic method for the presence or absence of antibody-response to a hepatitis B vaccine comprising the step of determining. Further, in the method, the polymorphic site of the DNA sequence of SEQ ID NO: 1 and SEQ ID NO: 3 (ie, 271 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1 and 311th of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 3 Base) appears as G and T as the labeling monobasic polymorphism (Tagging SNP), it may be determined that it belongs to the antibody-non-responsive group against the hepatitis B vaccine.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example

1. Subjects

The experiment was performed with 15 patients who were clinically diagnosed as antibody-no-responsive to the hepatitis B vaccine from the Korean tea hospital and 15 corresponding normal subjects. After the third dose of hepatitis antibody, hepatitis B surface antigen (HBsAg) test and Anti-HB (antibody against hepatitis B surface antigen) and titer were examined, which showed no response to the antigen, When no titer was less than 100U / ml, 15 patients were classified as antibody-non-responders and normal responders over 1000U / ml.

2. Preparation of Genomic DNA

To prepare genomic DNA (gDNA), blood was taken from antibody-unresponsive patients and 15 normal responders to 15 hepatitis B vaccines. 5 ml of total venous blood was obtained from the pre-vein of each volunteer and transferred to a Vacutainer tube containing heparin. Gentra's Blood genomic DNA prepartation kit was used to extract genomic DNA. The prepared genomic DNA was adjusted to a concentration of 750 ng / 15 ul using Quant-iT ^™ PicoGreen dsDNA reagent (Molecular Probes, Invitrogen) for Infinium ^™ Assay.

3. Genotyping of the Whole Genome-Infinium ^TM Assay

Described Infinium ^TM Assay is Sentrix ^R BeadChip (Illumina) the allele to one of the beads (bead) probes capable of distinguishing the (allele A and allele B) for each single nucleotide polymorphisms as microarrays used for Infinium ^TM Assay Planted. These probes consist of about 75 nucleotides, of which 25 nucleotides on the 5 'side are able to distinguish each single nucleotide polymorphism, while the remaining 50 nucleotides are designed to distinguish alleles for each single nucleotide polymorphism. have.

Amplification of the genome (Whole - genome amplification ( WGA )) Genomic DNA (750 ng / 15 ul) was dissolved at room temperature (22 ° C.) and transferred to each well of a 0.8 ml microtiter plate. 15 ul of 0.1 N NaOH was added and reacted at room temperature for 10 minutes. After the reaction, 270 ul of MP1 solution and 300 ul of AMM solution were added in sequence, and the mixture was turned over and mixed well. Centrifuged at 280 x g and reacted for 20 hours in a 37 ℃ oven.

The amplified fragment and purification of the genomic DNA the end of the total genomic DNA amplification step was separated one minutes and centrifuged at 50 × g, was transferred to 150 ul by three extra wells (well). As a result, four wells become the same genomic DNA. 50 ul of the FRG solution was added to each well, and then vortexed for 1 minute at 1600 rpm. After centrifugation at 50 x g at room temperature, the reaction was carried out at 37 ℃ for 1 hour. Genomic DNA was centrifuged at 50 xg, 100 ul of PA-1 solution was added and vortexed at 1600 rpm, centrifuged at 50 xg at room temperature, and then left at 37 ° C for 5 minutes. After centrifugation at 50 xg for 1 minute at room temperature, 300ul of 100% isopropanol was added and reacted at 4 ° C for 30 minutes. After centrifugation at 3000 xg for 20 minutes at 4 ° C and confirming genomic DNA clumps, the top layer was removed by inverting the microtiter plate. After drying the genomic DNA for 1 hour at room temperature, 42 ul RA1 solution was added, and reacted for 1 hour in a 48 ℃ oven.

Sentrix ^R Preparation of BeadChips and (Sentrix ^R ) BeadChips were prepared while resuspending the activated amplified genomic DNA in RA1 solution. The BeadChip was taken out and shaken up and down about 20 times in 100% ethanol and shaken with a reaction time interval of 5 minutes and 10 minutes. After the reaction in 100% ethanol, the BeadChip was shaken by transferring to PB1 solution, and shaken at the interval of 2.5 minutes and 5 minutes in the same manner as in 100% ethanol. After centrifugation at 280 xg for 1 minute at room temperature, it was assembled according to the recommended order (from Illumina) to prepare a BeadChip Chamber (called Flow through chamber). 150 ul of 100% formamide was passed through a BeadChip Chamber and 150 ul of RA1 solution was passed through twice. The BeadChip Chamber was placed in a Hybridization chamber containing PB2 solution until genomic DNA was added.

After resuspension of hybridized amplified genomic DNA (48 ° C., 1 hour reaction), vortex for 1 minute at 1800 rpm, centrifuge at 280 × g, and denature amplified genomic DNA for 20 minutes in a 95 ° C. heat block. The solution was centrifuged at 280 x g. Genomic DNA divided into four wells was pooled into one well and centrifuged at 280 × g. After removing the BeadChip Chamber from the Hybridization Chamber and adding genomic DNA, the BeadChip Chamber was placed in the Hybridization Chamber again and shaken at 48 ° C. for 18 hours to react.

After 18 hours of hybridization with Allele-Specific Primer Extension (ASPE), the BeadChip chamber was taken out and flowed 450 ul of RA1 solution four times. 450 ul of XB1 solution, 450 ul of XB2 solution, and 200 ul of EMM solution were sequentially reacted for 10 minutes, 5 minutes, and 15 minutes, respectively, and 450 ul of 95% formamide / 1 mM EDTA was flowed. 450 ul of XB3 solution was flowed and genomic DNA stained with LMM and ASM solution according to Illumina's protocol. After disassembling the BeadChip chamber, the BeadChip was washed with PB1 solution, dried by centrifugation at 280 × g for 1 minute, and then scanned using an Illumina BeadArray reader to store an image file for each BeadChip.

Genotyping Analysis of genotypes from image files was performed using Illuminas GenCall software (version 6.2.0.4) for genotyping for each monobasic polymorph. GenCall exhibits a typical genotype pattern with a percentage of the intensity of color development between probes recognizing two alleles and is reported as a GenCall score.

4. Statistical Analysis

Genotypes for each of the single nucleotide polymorphisms were analyzed using Haploview software (version 5.0), HapAnlayzer (version 1.1), and SNPAlyze (ver 5.1.1). First, from the results of each monobasic polymorphism, a small allele frequency (MAF)> 0.1 or above was used as a criterion, and HadAnalyzer conducted a Hardy-Weinberg Equilibrium (HWE) test to evaluate the significance level (p). single nucleotide polymorphs were selected. From the results selected, alleles and genotypes were analyzed for disease associations to determine the odds ratio (OR) for each and significant differences (<0.05) between the two groups from the significance level. ) Was examined. In addition, we conducted a linkage disequilibrium (LD) between single nucleotide polymorphisms that were closely related at the genetic level using Haploview (Fig. 1, Table 5). Associations were also tested (FIG. 2).

The chi-square test was used for the Heidi-Wineberg test and the haplotype association, and logistic regression was used for the analysis of risk and 95% confidence interval (CI). In addition, Lewontin's D 'and R ² measurements were used for the association of each polybasic polymorphism by the association equilibrium assay.

5. Observation on the analysis of monobasic polymorphs

From the results of each monobasic polymorphism, it can be seen that certain monobasic polymorphisms are closely related to each other and form a block unit, and these block units are called associative non-equilibrium blocks. It can be expected to move together during chromosome crossing over. It is also possible to determine the haplotype of the associative non equilibrium block by analyzing the observed genotypes (Fig. 1 and Table 5).

Table 5 shows the numerical value of the compactness of the associative non-equilibrium blocks of the single base polymorphisms shown in FIG. 1, and the lower part of the diagonal line represents the D 'value. When the value of D 'is 0.8 or more, it is determined that each of the monobasic polymorphisms has an associative non-equilibrium relationship and is shown in red as shown in FIG. The range of such D 'value is 0-1. In addition, the value of R ² represents the correlation between each polymorphic polymorphism. If the value of R ² is 0.3 or more, it means that the single polymorphisms corresponding to the value are closely packed. This means that in the aforementioned FIG. 1 and Table 5, the seven monobasic polymorphs are closely related to each other to form a genetic unit. In addition, these formed units are bundled together and transferred when passed to the next generation. Thus, non-equilibrium blocks appear to be patient-specific and cause disease, and if so affected, the block itself is passed on to the next generation, thus predicting the frequency of disease in the offspring of the next generation.

Genetic units formed from associative non-equilibrium blocks are determined from the genotyping of each monobasic polymorphism, which is called haplotype (FIG. 2).

FIG. 2 is a haplotype formed in comparison to genotyping results from the associative non equilibrium block formed from FIG. There are three haplotypes, the highest two of which are 'GCTGTAC' and 'AGCCCGG'. Since haplotypes are passed down to the next generation, it is necessary to analyze whether these haplotypes are associated with disease. Overall, disease-related analyzes of associative non-equilibrium blocks and haplotypes allow us to predict not only the diagnosis of the disease in the current generation, but also the transmission of the disease to the next generation. In addition, small inverted triangles are indicated in the first and third nucleotide sequences of each haplotype, indicating that the single base polymorphism present at the site represents the corresponding haplotype. For example, even if you do not analyze all the top haplotypes, if the single-base polymorphisms in the first and third positions are represented by G and T, the result can be judged as the highest haploid type. It means. Such single nucleotide polymorphisms that can represent haplotypes are called tagging SNPs.

Haplotype Case: freq Cont: freq Hvs- Hvs- Hvs- OR (95% c.i.) Chi-square p-value HAP1 GCAGTAC 0.67 0.3 4.67 (1.57-13.87) 6.67 0.0098 HAP2 AGGCCGG 0.23 0.33 0.61 (0.20-1.90) 0.33 0.5667 HAP3 AGAGCGC 0.1 0.37 0.19 (0.05-0.78) 4.57 0.0326

Table 6 shows the results of the chi-square test showing whether the three types of haplotypes determined from associative non-equilibrium blocks are significantly different in the antibody-non-responsive and normal-response groups against the hepatitis B vaccine. From these results, it can be seen that the haplotype of the uppermost group was significantly higher (p value <0.0098) in the normal group.

In view of the results of such an analysis, each allele may exist in the form of a homozygote or heterozygote in each individual. Mendel's genetic law and Hardy-Weinberg law show that the composition of the alleles that make up a group is maintained at a constant frequency and, if statistically significant, can give a biological function meaning. The monobasic polymorphisms of the present invention appear at statistically significant levels in antibody-unresponsive patients to hepatitis B vaccines, as well as the diagnosis of antibody-response to hepatitis B vaccines from these monobasic polymorphisms to genetic units. It can be seen that it can be used for prediction.

The polynucleotides or complementary nucleotides of at least one selected from the group consisting of polynucleotides (b), (c), (f) and (g) according to the invention are useful for the diagnosis of antibody-response to hepatitis B vaccine. Can be used.

In addition, primers or probes consisting of the polynucleotide or its complementary nucleotides, and the polynucleotide or the complementary nucleotide kit thereof, can be usefully used for the diagnosis of antibody-nonresponse against hepatitis B vaccine.

In addition, the diagnostic method of the present invention can effectively diagnose the antibody-no response to the hepatitis B vaccine.

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Claims (13)

delete delete delete In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more contiguous DNA sequences comprising the 218th base of G and the 218th base (polymorphic site); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, the polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 311 th base (polymorphic site) is C and comprising the 311 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, a polynucleotide (f) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) C and comprising the 301th base; And In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide consisting of at least 10 consecutive DNA sequences comprising the 294th base of which the 294th base (polymorphic site) is A and (g) Amplification primers for antibody-non-response diagnosis against hepatitis B vaccine, consisting of at least one polynucleotide selected from the group consisting of or complementary nucleotides thereof. In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more contiguous DNA sequences comprising the 218th base of G and the 218th base (polymorphic site); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, the polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 311 th base (polymorphic site) is C and comprising the 311 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, a polynucleotide (f) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) C and comprising the 301th base; And In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide consisting of at least 10 consecutive DNA sequences comprising the 294th base of which the 294th base (polymorphic site) is A and (g) An antibody-non-response diagnostic probe for a hepatitis B vaccine consisting of at least one polynucleotide selected from the group consisting of or complementary nucleotides thereof. Microarray comprising an antibody-non-response diagnostic probe for hepatitis B vaccine according to claim 5. In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more contiguous DNA sequences comprising the 218th base of G and the 218th base (polymorphic site); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, the polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 311 th base (polymorphic site) is C and comprising the 311 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, a polynucleotide (f) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) C and comprising the 301th base; And In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide consisting of at least 10 consecutive DNA sequences comprising the 294th base of which the 294th base (polymorphic site) is A and (g) A kit for antibody-non-response diagnostics for hepatitis B vaccine comprising at least one polynucleotide selected from the group consisting of or complementary nucleotides thereof. Obtaining a nucleic acid sample from the sample; And Determining the nucleotide sequence of one or more polymorphic sites of the polynucleotides of SEQ ID NOs: 2, 3, 6, and 7 or complementary nucleotides thereof, wherein the polymorphic sites are as defined in claim 4 Antibody-response diagnostic method for hepatitis B vaccine comprising a. The method of claim 8, wherein determining the nucleotide sequence of the polymorphic site Hybridizing the nucleic acid sample to a microarray to which a polynucleotide selected from the group consisting of polynucleotides (b), (c), (f) and (g) or a complementary nucleotide thereof is immobilized, provided that the polynucleotide (b ), (c), (f) and (g) as defined in claim 4); And Detecting the obtained hybridization results Diagnostic method comprising a. The method according to claim 8, wherein when the nucleotide sequence of the polymorphic site is determined, when one or more bases of the polymorphic sites of SEQ ID NOs: 2, 3, 6, and 7 are G, C, C, and A, respectively, B is A diagnostic method characterized in that it is determined to belong to the antibody-non-responsive group to the hepatitis vaccine. The method according to claim 8, wherein the nucleotide sequence of the polymorphic site is determined, the dominant genotype of the polymorphic site of SEQ ID NO: 2, 3, 6, and 7 is G / G, G / A, C / C and A diagnostic method characterized in that it is determined to belong to the antibody-non-responsive group to the hepatitis B vaccine when at least one of C / T, C / C and C / G, and A / A and A / G is present . Obtaining a nucleic acid sample from the sample; And 271 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1; 218th base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2; 311 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 3; 301th base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 4; 501st base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 5; Base 301 of the polynucleotide having a DNA sequence of SEQ ID NO: 6; And if the haplotype analyzed from the 294th base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 7 is GCTGTAC, determining that it belongs to the antibody-non-responsive group against the hepatitis B vaccine. Method for diagnosing the presence or absence of antibody-no response to hepatitis vaccine. Obtaining a nucleic acid sample from the sample; And Hepatitis B vaccine when the 271 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1 and the 311 base of the polynucleotide consisting of the DNA sequence of SEQ ID NO: 3 appear as G and T as the tagging SNPs A method for diagnosing the presence or absence of an antibody-response to a hepatitis B vaccine comprising the step of determining that the antibody-response group belongs to.

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