JP2007228941A - Gene associated with presence or absence of susceptibility to acute exacerbation in chronic obstructive pulmonary disease and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for predicting and judging whether or not susceptibility to acute exacerbation is present in COPD (chronic obstructive pulmonary disease) using a gene associated with the susceptibility to acute respiratory tract infections (acute exacerbation) in the COPD or polymorphisms associated with the susceptibility to the acute respiratory tract infections (acute exacerbation) in the COPD existing in a near DNA region of the gene as an index. <P>SOLUTION: An analysis of 200 SNPs (single nucleotide polymorphisms) around 94 genes associated with immunity and inflammation considered to be associated with general infections is carried out from nucleic acid database search about genetic diathesis which is a risk factor of the susceptibility to the acute respiratory tract infections (acute exacerbation) in patients suffering from the COPD. Thereby, it is verified whether or not the susceptibility to the acute respiratory tract infections (acute exacerbation) in the patients suffering from the COPD is caused by the conversion of the CCL1 (chemokine cc motif ligand 1) gene produced from the SNPs. When a mutation in base species in a polymorphic site is detected as in the case of the SNP rs2282691 (ID: NCBI SNP reference), the susceptibility to the acute exacerbation is judged to be present in the patients suffering from the COPD. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene associated with the presence or absence of susceptibility to acute exacerbation (hereinafter also referred to as AECOPD) in chronic obstructive pulmonary disease (hereinafter referred to as COPD), and the gene or the gene It relates to polymorphisms related to the presence or absence of AECOPD susceptibility, which are present in the vicinity DNA. The present invention also relates to a method for determining whether or not AECOPD using the polymorphism comes frequently, or determining the severity (mortality) of AECOPD. Furthermore, a screening method for AECOPD therapeutic or prophylactic agents is also provided.

  COPD occupies a high position in morbidity and mortality worldwide. In a recent review of The global burden of human illness, COPD is ranked 12th as a disease that reduces mortality and quality of life, and is ranked 3rd in mortality by 2020 and reduces quality of life. It is predicted to rise to fifth place as a chronic disease to be caused (Non-Patent Documents 1 and 2).

  Acute COPD exacerbation (AECOPD) is a common cause of disease deterioration in COPD patients (Non-Patent Documents 3 and 4) and the most common cause of death (Non-Patent Documents 5 and 6), and also for medical resources It is a huge burden. AECOPD is now recognized as having serious consequences for both individuals and society. According to the assessment of health surveys with an emphasis on quality of life, the adverse effects of the disease for such patients are significantly higher in the high frequency group of acute hatred than in the low group. Even if acute hatred is not enough to receive treatment in a hospital, it has been proved to be the same result (Non-patent Document 7). In addition, hospitalization for AECOPD is also the most expensive part of COPD treatment. The number of hospitalizations due to AECOPD has been on the rise in recent years, reflecting the increase in the elderly population, leading to a shortage of hospitalized bets, especially in winter, and causing the Japanese medical system to be exhausted.

  Infection accounts for most of the causes of acute hatred (AECOPD) in COPD. Only a few of the causes of AECOPD are caused by pulmonary heart. Therefore, it can be considered that acute respiratory tract infection in COPD is practically synonymous with AECOPD.

  AECOPD is considered to be a disease composed of various elements in the underlying mechanism of progress. Among them, host defense factors against pathogens such as bacteria and viruses are important for repetitive AECOPD for the following reasons. It is suggested that it plays a role. First of all, the cellular immune response and the humoral immune response to the causative pathogens vary from patient to patient. Such responsiveness of cellular immunity is associated with the occurrence of AECOPD (Non-patent Documents 8 and 9). Secondly, 25-50% of COPD patients have a chronic flora of pathogenic bacteria constantly formed in the lower respiratory tract, indicating that the host defense mechanism is broken. It has been suggested that the presence of such pathogenic bacterial flora in the lower respiratory tract of COPD patients may affect both the frequency and severity of AECOPD and the phenotype of the disease state (Non-patent Document 10). . Third, although there is a correlation between the frequency of AECOPD and the amount of inflammatory cytokines such as IL-6, IL-8, etc. (Non-Patent Document 11) in sputum, inflammatory properties throughout the period of AECOPD There is considerable diversity in the amount of markers, suggesting that this forms a variety of inflammatory responses in AECOPD (Non-Patent Documents 11, 12, and 13).

  In managing the risk of developing AECOPD in COPD patients, it is very useful to have a biomarker that can determine the risk before the onset, rather than a marker that changes after the onset of AECOPD. As classics, age, BMI, pulmonary function (especially the predicted amount per second) and the like have been conventionally known as AECOPD risk factors (Non-patent Document 14). In addition, although there is a correlation between inflammatory cytokines such as IL-6 and IL-8 in the induced sputum and the frequency of AECOPD when viewed as a certain number of patient populations (Non-patent Document 11), cytokines There is considerable variation in the baseline amount of individuals, and it is difficult to use as a biomarker to determine the individual's risk of developing AECOPD.

  For the reasons described above, it is possible that at least part of an individual's susceptibility to AECOPD is genetic, which may contribute to the diversity characteristics of AECOPD, and thus such genetic Testing genes for AECOPD susceptibility may be extremely useful in determining individual patient treatment strategies. In fact, several long-term cohort studies have shown that AECOPD has a wide range of onset frequencies (Non-Patent Documents 15, 16, and 17). If a patient having a genetic risk factor of acute hatred can be determined in advance, the patient's disease development progress and prognosis can be predicted, which is very useful in treatment. The inventors have conducted extensive research to identify new genetic risk factors for acute COPD hatred (AECOPD).

  The gene discovered by the inventors as a new genetic risk factor is chemokine CC motif ligand 1 (CCL1). Chemokines are a group of cytokines having leukocyte chemotaxis and a molecular weight of 8 to 10 kDa, and are classified into four subfamilies based on the structure (Non-patent Documents 18 and 19). The chemokine subfamily constitutes a family that includes mediators of inflammation and immunity, particularly regulation of immune defense and housekeeping of the immune system (Non-Patent Documents 18 and 19). Inflammatory chemokines are secreted proteins produced by many tissue cells and migrate leukocytes in response to bacterial toxins and inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ. The effect is local, and there are both paracrine and autocrine forms (Non-patent Documents 18 and 19). In addition to the function of migrating leukocytes, it also plays a central role in immune regulation (both T cell activation and antigen presentation function), enhancing antigen-specific helper T1 (Th1), activating Th2, It also plays a role in promoting cell division and release of various lymphokines (Non-patent Documents 18 and 19).

  Among chemokines, CCL11 and CCL5 are both representative eosinophil chemotactic factors and have been reported to play an important role in AECOPD in stable mild COPD (Non-patent Documents 20 and 21). . Pathologically, COPD is characterized as chronic inflammation and remodeling that occurs in all of the airways, parenchyma, and pulmonary vasculature (22-25). Stable COPD is characterized by increased infiltration of the tracheal mucosa by increased CD8 + T lymphocytes, monocytes and macrophages (Non-patent Documents 26-29). However, in the acute exacerbation of mild COPD, there is an accumulation of eosinophils in the mucosa, which is two typical eosinophil migration factors that are strongly expressed in airway epithelium and subepithelial lymphocytes and monocytes. According to up-regulation of Eotaxin (CCL11) and RANTES (CCL5) (Non-patent Documents 20 and 21). It has been reported that this “allergic” characteristic in airway inflammation in mild COPD acute exacerbations is replaced by an increase in the number of neutrophils in the airway in acute exacerbations of COPD.

  CXCL5 and CXCL8 are both typical neutrophil chemotactic factors, but they are associated with moderate to severe COPD acute exacerbation, and neutrophil proliferation into the airways, a hallmark of exacerbation Although it has been reported to play a central role in many cases (Non-patent Document 30), there is no known association between CCL1 and AECOPD, particularly susceptibility to AECOPD in COPD patients. In addition, the frequency of hatred increases in proportion to the severity of COPD (Non-patent Document 31).

Thus, although there are some reports on the relationship between other chemokines and inflammation of the airway mucosa, the inventors have discovered a new genetic risk factor, a gene specific to monocytes. It is not known that CCL1 (Non-patent Document 32) encoding a gene known as a chemotactic factor is associated with the frequency or severity of AECOPD.
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  The present invention has been made in view of such circumstances, and the object thereof is present in a gene related to susceptibility to acute exacerbation (AECOPD) in COPD, and the gene or a DNA region adjacent to the gene. To find polymorphisms associated with AECOPD susceptibility.

  It is another object of the present invention to provide a method for predicting whether or not AECOPD is susceptible using the polymorphism related to AECOPD susceptibility existing in the gene or a DNA region adjacent to the gene as an index.

  In order to solve the above problems, the present inventors used genomic DNA from 276 male COPD patients to search for genes responsible for acute exacerbation (AECOPD) susceptibility in COPD patients. By analyzing 200 single nucleotide polymorphisms (SNPs) of 94 genes related to immunity / inflammation, susceptibility to AECOPD has been increased by chemokine CC motif ligand 1 (CCL1) Found related to. In other words, the inventors found that SNP rs2282691 on the CCL1 gene is a sensitive SNP related to the incidence of AECOPD susceptibility or the severity (mortality) of infection, and the susceptibility to infection in COPD patients. The inventors found that the susceptibility is attributed to a polymorphism of the CCL1 gene, thereby completing the present invention.

More specifically, the present invention provides the following (1) to (19).
(1) A method for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, which comprises detecting a mutation in the CCL1 gene or a nearby DNA region of the gene in a subject.
(2) The method according to claim 1, wherein it is determined that the mutation is susceptible to acute exacerbation in chronic obstructive pulmonary disease when it results in a decrease in the expression level of the gene.
(3) The method according to claim 1 or 2, wherein the mutation is a single nucleotide polymorphism mutation.
(4) A method for determining the presence or absence of susceptibility to acute exacerbation in chronic obstructive pulmonary disease for a subject, comprising the following steps (a) and (b).
(A) A step of determining a base type of a polymorphic site in a CCL1 gene or a DNA region adjacent to the gene in a subject.
(B) A step of determining that the subject is susceptible to acute exacerbation in chronic obstructive pulmonary disease when a mutation is detected in the base type of the polymorphic site determined in (a).
(5) The method according to claim 4, wherein it is determined that the mutation is susceptible to acute exacerbation in chronic obstructive pulmonary disease when it results in a decrease in the expression level of the gene.
(6) The method according to claim 4 or 5, wherein the polymorphic site is a site on the CCL1 gene region and is a polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1.
(7) The base type mutation of claim 4 to 6, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 1 is mutated from T to A at a site on the CCL1 gene region. The method according to any one.
(8) The method according to any one of claims 1 to 7, wherein a biological sample derived from a subject is used as a test sample.
(9) The method according to any one of claims 1 to 8, wherein the susceptibility to acute exacerbation in chronic obstructive pulmonary disease is the frequency of acute exacerbation in chronic obstructive pulmonary disease.
(10) The method according to any one of claims 1 to 8, wherein the susceptibility to acute exacerbation in chronic obstructive pulmonary disease is the severity of acute exacerbation in chronic obstructive pulmonary disease.
(11) The method according to any one of claims 1 to 10, wherein the acute exacerbation in chronic obstructive pulmonary disease is an acute respiratory tract infection in chronic obstructive pulmonary disease.
(12) To determine the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to the DNA comprising the polymorphic site according to claim 7 Drugs.
(13) A drug for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising a solid phase to which a nucleotide probe that hybridizes with DNA containing the polymorphic site according to claim 7 is immobilized.
(14) A drug for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising a primer oligonucleotide for amplifying the DNA comprising the polymorphic site according to claim 7.
(15) The drug according to any one of claims 12 to 14, wherein the acute exacerbation in chronic obstructive pulmonary disease is an acute respiratory tract infection in chronic obstructive pulmonary disease.
(16) A screening method for a therapeutic or preventive agent for acute exacerbation in chronic obstructive pulmonary disease, comprising selecting a substance that activates the expression level of the CCL1 gene or the function of a protein encoded by the gene.
(17) A screening method for a therapeutic or prophylactic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising the following steps (a) to (c):
(A) a step of bringing a test substance into contact with a cell expressing the CCL1 gene; (b) a step of measuring the expression level of the CCL1 gene; and (c) the expression compared to a case where the test substance is measured in the absence of the test substance. (18) A method for screening a therapeutic or preventive agent for acute exacerbation in chronic obstructive pulmonary disease, comprising the step (18) of selecting a substance that increases the level:
(A) a step of bringing a test substance into contact with a cell or a cell extract containing DNA having a structure in which a transcriptional regulatory region of the CCL1 gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene Step (c) Step (19) of selecting a substance that increases the expression level as compared with the case where it is measured in the absence of the test substance (19) Chronic obstructiveness comprising the following steps (a) to (c) A screening method for a therapeutic or prophylactic agent for acute exacerbations in lung diseases.
(A) contacting a polynucleotide containing a C / EBPβ recognition sequence and C / EBPβ protein with a test substance (b) measuring a binding activity between the polynucleotide and a transcription factor (c) absence of the test substance The screening according to any one of claims 16 to 19, wherein the step (20) of selecting a substance that enhances the binding activity as compared to the case measured below is an acute respiratory tract infection in acute obstructive pulmonary disease Method.
(21) A prophylactic or therapeutic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising as an active ingredient a substance that activates the expression level of the CCL1 gene or the function of the protein encoded by the gene.

  In accordance with the present invention, the frequency and severity of AECOPD by detecting mutations in the gene associated with acute exacerbations in COPD (AECOPD) and in DNA regions on or near the gene (herein A method for determining AECOPD susceptibility) was provided. By detecting a polymorphism on the gene of the present invention or in a DNA region in the vicinity of the gene, whether or not it is susceptible to subsequent AECOPD in the COPD patient, and whether or not a more serious respiratory tract infection is caused Becomes predictable. The present invention makes it possible to establish a more efficient strategy (so-called tailor-made medicine) in the prevention and treatment of respiratory tract infections in COPD. Specifically, by diagnosing susceptibility to AECOPD, patients who have been determined to be susceptible to AECOPD are actively vaccinated with pneumococcal vaccine in addition to influenza vaccine, prescription of expectorants, prophylactic antibiotics It is very promising because it will help to determine treatment policy such as prescription of substances, and may lead to the development of new therapeutic agents that prevent AECOPD.

  The inventors have identified genes and polymorphisms associated with susceptibility to acute exacerbations in COPD (AECOPD). In COPD patients who are susceptible to AECOPD, mutations are significantly found in this gene or in the vicinity DNA region of this gene. Diagnosis of AECOPD susceptibility to the elderly.

    In the present invention, “susceptibility to acute exacerbation in COPD (AECOPD)” means that the frequency of suffering from acute aversion (AECOPD) in a COPD patient and the fatality rate due to an infectious disease are high. Compared to normal, AECOPD is judged comprehensively in addition to increased sputum volume, color tone, and viscosity, as well as increased dyspnea, increased cough, fever, and general condition deterioration. COPD hatred.

The gene found by the present inventors to be associated with AECOPD is the CCL1 gene (GenBank accession number: NM_002981).
Information on the base sequence of this gene and the amino acid sequence of the protein encoded by this gene can be easily obtained from the above-mentioned GenBank accession number. Moreover, those skilled in the art can easily obtain information on the base sequence of a gene and the amino acid sequence of a protein encoded by the gene from a public gene database or literature database based on the above gene notation (gene name). It is possible.

  Based on the above findings, the present invention provides a method for determining susceptibility to AECOPD, comprising detecting a mutation in the CCL1 gene or a nearby DNA region of the gene in a subject.

In the present invention, “determining susceptibility to acute exacerbation (AECOPD) in COPD” means that it is more or less likely that the subject's COPD patient will suffer acute respiratory tract infection more than once in two years, and Includes testing to determine if an individual has an acute respiratory tract infection, develops a serious infection, and is more likely to die. In the method of the present invention, when a mutation is detected in the CCL1 gene or a nearby DNA region of the gene, it is determined that the subject is susceptible to AECOPD or has a predisposition to susceptibility to AECOPD.
On the other hand, if no mutation is detected in the CCL1 gene or a DNA region adjacent to the CCL1 gene, the subject is determined to be resistant or predisposed to resistance to AECOPD.

  According to the method of the present invention, even a subject who does not suffer from COPD can determine the susceptibility to AECOPD when suffering from COPD. Further, by determining the susceptibility of AECOPD in the case of a subject who already suffers from COPD, a more effective or efficient treatment strategy (so-called customized medicine) can be established. Specifically, for patients who are determined to be susceptible to AECODP, treatment policies such as active inoculation of pneumococcal vaccine in addition to influenza vaccine, prescription of expectorants, prescription of prophylactic antibiotics, etc. It can be used for etc.

  As used herein, “treatment” generally means obtaining pharmacological and / or physiological effects. The effect may be preventive in that the disease or symptom is completely or partially prevented, or may be therapeutic in that the symptom of the disease is completely or partially treated. “Treatment” as used herein includes all treatment of diseases in mammals, particularly humans. In addition, "treatment" includes prevention of onset, suppression of disease progression, or reduction of disease in subjects who are predisposed to the disease but have not yet been diagnosed. .

  Specific examples of the DNA sequence of the CCL1 gene of the present invention and the DNA sequence in the vicinity of the gene include the sequence described in SEQ ID NO: 1, for example. The sequence described in SEQ ID NO: 1 is a sequence prepared based on the result of mapping info (NCBI build34). In the present invention, the “near DNA region of a gene” usually refers to a region on a chromosome in the vicinity of the gene. The vicinity is not particularly limited, but is usually a DNA region containing the polymorphic site of the present invention.

  Although the position of the “mutation” in the test method of the present invention is difficult to predefine, it is usually in the ORF of the gene or a region that controls the expression of the gene (eg, promoter region, enhancer region, etc.) ), But is not limited thereto. In addition, the “mutation” is a mutation that changes the expression level of the gene, changes properties such as mRNA stability, or changes the activity of the protein encoded by the gene. Many, but not particularly limited. Examples of the mutation of the present invention include base addition, deletion, substitution, insertion mutation and the like.

  The present inventors have succeeded in finding a polymorphic mutation significantly related to susceptibility to AECOPD in the CCL1 gene of the present invention or a nearby DNA region of the gene in a subject. Therefore, by using the presence or absence of mutation as an index (determining the base type) for the polymorphic site on the CCL1 gene of the present invention or a DNA region adjacent to the gene, it is possible to test whether or not it is susceptible to AECOPD. Is possible.

The “near DNA region of the gene” mentioned above usually refers to a region on the chromosome in the vicinity of the gene. Although the neighborhood is not particularly limited, it is usually a DNA region containing the polymorphic site of the present invention, preferably from the polymorphic site or the end site of the LD block (linkage disequilibrium block) containing the polymorphic site. Refers to a region within 10 kb.
As reported by Gabriel et al., Polymorphisms in the range of 10 kb before and after, that is, within 20 kb are likely to be linked (Gabriel SB, Schaffner SF, Nguyen H et al. The structure of haplotype blocks in the human genome. Science 296, 2225-9. 2002).

  An example of the CCL1 gene and a DNA sequence in the vicinity of the gene is shown in SEQ ID NO: 1.

  In a preferred embodiment of the present invention, whether or not it is susceptible to AECOPD, characterized by detecting a polymorphic mutation (base species at the polymorphic site) in the CCL1 gene of the present invention or a nearby DNA region of the gene. It is a method of inspection.

  A polymorphism is generally defined genetically as a change in a base in one gene that is present at a frequency of 1% or more in the population. Not limited to. Examples of the polymorphism in the present invention include a single nucleotide polymorphism and a polymorphism in which one to several tens of bases (sometimes several thousand bases) are deleted or inserted. Furthermore, the number of polymorphic sites is not limited to one, and may be a plurality of polymorphs.

The present invention also provides a method for examining susceptibility to AECOPD, characterized by determining the base type of the polymorphic site in the CCL1 gene of the present invention or a DNA region in the vicinity of the subject.
The “polymorphic site” determined in the method of the present invention is not particularly limited as long as it is a polymorphism present in the CCL1 gene of the present invention or a DNA region adjacent to the gene. Specifically, as a polymorphic site that can be used in the AECOPD susceptibility testing method, the CCL1 gene or a site on a DNA region in the vicinity of the gene, which is a polymorphism at position 10001 in the base sequence described in SEQ ID NO: 1 A mold site can be mentioned. (In the present specification, these polymorphic sites may be simply referred to as “polymorphic sites of the present invention”).

Abbreviation definition: NCBI = National Center for Biotechnology Information;
CCL1 = Chemokine CC motif ligand 1

  Moreover, those skilled in the art can appropriately acquire information on the base type for the site based on the rs number in the published dbSNP database. In addition, in the NCBI SNP reference column, those with rs at the beginning are given IDs that are uniquely determined by NCBI among the registration IDs in the dbSNP database. The dbSNP database is published on the website (http://www.ncbi.nlm.nih.gov/SNP/index.html), and the website is registered using the registration ID number described in the NCBI SNP reference column. By performing the above search, detailed information on the SNPs in the base sequence (for example, the position on the chromosome, the type of base at the polymorphic site, the sequence before and after, etc.) can be obtained. When such information is used, those skilled in the art can easily perform the inspection described in the present invention. As for information on the CCL1 polymorphic site, Tables 1A to 1C show information on the polymorphic site present at 10 kb around the polymorphism at position 10001 in Sequence Listing 1.

  The base type of the polymorphic site shown in Tables 1A to C may indicate a base type that is on the complementary strand side with respect to the sequence shown in the sequence listing, but the front and back sequences described in this specification, or It is easy for those skilled in the art to confirm the difference by using the sequence before and after published in the dbSNP and JSNP databases, and it is inevitably necessary to examine either the plus strand or the minus strand when conducting the test. The result can be determined. As for the human genome sequence, the human genome international project build35, which is said to be almost the final version, has been announced. The sequences described in this specification are based on the results of the human genome international project build34. However, those skilled in the art can easily substitute the information in the build 35 from the information in the sequence listing attached to the present specification.

  In addition, those skilled in the art usually use the registration ID number assigned to the polymorphism disclosed in the present specification, for example, the rs number in the dbSNP database, to determine the actual genomic position of the polymorphic site of the present invention and The arrangement and the like can be easily known. Thus, even if it is not possible to know, the person skilled in the art can appropriately obtain information on the actual genome corresponding to the polymorphic site from the information on the base sequence and polymorphic site shown in SEQ ID NO: 1. It is easy to know the location. For example, the position of the polymorphic site of the present invention on the genome can be known by making an inquiry with a publicly available genome database or the like. That is, even if a slight base sequence difference is observed between the base sequence described in the sequence listing and the actual base sequence on the genome, By performing a homology search or the like, it is possible to accurately know the actual genomic position of the polymorphic site of the present invention. Even when the position on the genome cannot be specified, it is easy to perform the test described in the present invention from the sequence listing and polymorphic site information described in this specification.

  Genomic DNA usually has double-stranded DNA structures complementary to each other. Therefore, in the present specification, even when a DNA sequence in one strand is shown for convenience, it is understood that a sequence complementary to the sequence (base) is also disclosed as a matter of course. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious.

  In the method for testing susceptibility to AECOPD in COPD of the present invention, the base species of the polymorphic site at position 10001 in the CCL1 gene or a site on the DNA region in the vicinity of the gene and in the base sequence set forth in SEQ ID NO: 1 is used. It is preferable to make this determination.

  In a preferred embodiment of the present invention, the base species at position 10001 in the base sequence described in SEQ ID NO: 1 is A (hetero and homo) in the CCL1 gene of the present invention or a site on the DNA region adjacent to the gene. Is determined to be susceptible to AECOPD.

  In the present invention, it is considered that the polymorphic site and its surrounding DNA region are strongly linked even if other than the polymorphic site. It is also possible to test for susceptibility to infection in COPD by determining the species. That is, this `` for a small population of humans including patients with frequent AECOPD or those who developed serious infections leading to death in which the base type of the polymorphic site is the above base type '' The base species in the “near polymorphic site” (for example, the polymorphic site described in Table 1 above) is determined in advance.

  Next, a base type in the subject is determined for this “neighboring polymorphic site” and compared with the base type determined in advance, an AECOPD susceptibility test can be performed. A subject is determined to be susceptible to AECOPD when the base type is the same as the predetermined base type. According to the test method of the present invention, it is possible to determine whether or not a COPD patient is susceptible to AECOPD, and it can be used for determination of a treatment policy, determination of a drug dose, and the like.

  For a small population of humans including AECOPD-susceptible patients whose base type of the polymorphic site at position 10001 in the base sequence set forth in SEQ ID NO: 1 on the CCL1 gene is A, a nearby polymorphic site, for example, position 10043 The base type of the polymorphic site is determined. If the base species at this site is known to be susceptible to AECOPD, the frequency of G is higher than that of those known to be not susceptible to AECOPD in COPD. When the base type of the polymorphic site at position 10043 is examined for the examiner and the base type at this site is also G, the subject is determined to be susceptible to infection in COPD.

  As described above, according to the present invention, the region on the gene related to AECOPD susceptibility has been clarified, so that whether or not AECOPD is susceptible to AECOPD in the above-mentioned COPD without imposing an excessive burden on those skilled in the art. Inspection can be performed.

  A person skilled in the art can determine the base species in the polymorphic site of the present invention by various methods. For example, it can be performed by directly determining the base sequence of DNA containing the polymorphic site of the present invention.

In general, the test sample used in the test method of the present invention is preferably a biological sample obtained in advance from a subject. Examples of the biological sample include a DNA sample. The DNA sample in the present invention is based on, for example, a subject's blood, skin, oral mucosa, tissue or cells collected or excised by surgery, chromosomal DNA extracted from body fluid collected for the purpose of examination, or RNA. Can be prepared.
That is, the present invention is a method in which a biological sample derived from a subject (a biological sample obtained in advance from the subject) is usually used for the examination as a test sample.

A person skilled in the art can appropriately prepare a biological sample using a known technique. For example, a DNA sample can be prepared by PCR using a primer that hybridizes to DNA containing the polymorphic site of the present invention and chromosomal DNA or RNA as a template.
In this method, the base sequence of the isolated DNA is then determined. Those skilled in the art can easily determine the base sequence of the isolated DNA using a DNA sequencer or the like.

  In the polymorphic site of the present invention, usually the variation of the base type of the site has already been clarified. “Determining the base type” in the present invention does not necessarily mean that the polymorphic site is a base type of A, G, T, or C. For example, if it is known that the variation of the base species is A or G for a certain polymorphic site, it is sufficient that the base species at that site is found to be “not A” or “not G”. .

  Various methods for determining the base type of a polymorphic site whose base variation has been clarified in advance are known. The method for determining the base species of the present invention is not particularly limited. For example, TaqMan PCR method, AcycloPrime method, MALDI-TOF / MS method and the like have been put to practical use as analysis methods applying the PCR method. Invader and RCA methods are known as methods for determining base types that do not depend on PCR. Furthermore, the base species can be determined using a DNA array. Some methods are briefly described below. Any of the methods described herein can be applied to the determination of the base type of the polymorphic site in the present invention, but the method is not limited.

[TaqMan PCR method]
The principle of TaqMan PCR method is as follows. The TaqMan PCR method is an analysis method using a primer set that can amplify an allele-containing region and a TaqMan probe. The TaqMan probe is designed to hybridize to the allele-containing region amplified by this primer set.

  If the target base sequence is hybridized under conditions close to the Tm of the TaqMan probe, the hybridization efficiency of the TaqMan probe is significantly reduced due to the difference of one base. When PCR is performed in the presence of the TaqMan probe, the extension reaction from the primer eventually reaches the hybridized TaqMan probe. At this time, the TaqMan probe is degraded from its 5 ′ end by the 5′-3 ′ exonuclease activity of DNA polymerase. If the TaqMan probe is labeled with a reporter dye and a quencher, the degradation of the TaqMan probe can be followed as a change in fluorescence signal. In other words, when the TaqMan probe is decomposed, the reporter dye is released and the distance from the quencher is increased to generate a fluorescent signal. If the hybridization of the TaqMan probe decreases due to a difference in one base, the TaqMan probe does not decompose and a fluorescent signal is not generated.

  If a TaqMan probe corresponding to a polymorphism is designed and a different signal is generated by the decomposition of each probe, the base species can be determined at the same time. For example, as a reporter dye, 6-carboxy-fluorescein (FAM) is used for an allyl A TaqMan probe of an allyl and VIC is used for an allyl B probe. In a state where the probe is not decomposed, generation of a fluorescent signal of the reporter dye is suppressed by the quencher. If each probe hybridizes to the corresponding allele, a fluorescence signal corresponding to the hybridization is observed. That is, when either FAM or VIC signal is stronger than the other, it turns out that allyl A or allyl B is homozygous. On the other hand, when allyl is hetero, both signals are detected at almost the same level. By using the TaqMan PCR method, PCR and base type determination can be performed simultaneously on a genome as an analysis target without a time-consuming step such as separation on a gel. Therefore, the TaqMan PCR method is useful as a method that can determine the base species for many subjects.

[AcycloPrime method]
The AcycloPrime method has also been put into practical use as a method for determining the base species using the PCR method. In the AcycloPrime method, one set of primers for genome amplification and one set of primers for polymorphism detection are used. First, a region containing a polymorphic site in the genome is amplified by PCR. This process is the same as normal genomic PCR. Next, the obtained PCR product is annealed with a primer for detecting SNPs, and an extension reaction is performed. The primer for detecting SNPs is designed to anneal to a region adjacent to the polymorphic site to be detected.
At this time, a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocked with 3′-OH is used as a nucleotide substrate for the extension reaction. As a result, only one base complementary to the base at the position corresponding to the polymorphic site is incorporated, and the extension reaction stops. Incorporation of a nucleotide derivative into a primer can be detected by an increase in fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels having different wavelengths are used for the fluorescent polarizing dye, it is possible to specify which of the two types of bases the specific SNPs are. Since the level of fluorescence polarization can be quantified, it is also possible to determine whether allyl is homo or hetero in one analysis.

[MALDI-TOF / MS method]
The base species can also be determined by analyzing the PCR product with MALDI-TOF / MS. MALDI-TOF / MS can be used in various fields as an analytical method that can clearly identify slight differences in amino acid sequences of proteins and DNA base sequences because it can know the molecular weight with great accuracy. Yes. In order to determine the base species by MALDI-TOF / MS, first, the region containing allyl to be analyzed is amplified by PCR. The amplification product is then isolated and its molecular weight is measured by MALDI-TOF / MS. Since the base sequence of allyl is known in advance, the base sequence of the amplification product is uniquely determined based on the molecular weight.
In order to determine the base species using MALDI-TOF / MS, a PCR product separation step is required. However, accurate base species determination can be expected without using labeled primers or labeled probes. It can also be applied to simultaneous detection of polymorphisms at multiple locations.

[SNPs-specific labeling method using type IIs restriction enzyme]
A method that can determine the base species at higher speed using the PCR method has also been reported. For example, the base type of a polymorphic site is determined using a type IIs restriction enzyme. In this method, a primer having a recognition sequence for type IIs restriction enzyme is used for PCR. A general restriction enzyme (type II) used for gene recombination recognizes a specific base sequence and cleaves a specific site in the base sequence. In contrast, type IIs restriction enzymes recognize specific base sequences and cleave sites away from the recognized base sequences. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme. Therefore, if the primer containing the recognition sequence of the type IIs restriction enzyme is annealed at a position separated by the number of bases, the amplified product can be cleaved at the polymorphic site by the type IIs restriction enzyme.

  At the end of the amplification product cleaved with the type IIs restriction enzyme, a cohesive end containing the base of SNPs is formed. Here, an adapter having a base sequence corresponding to the sticky end of the amplification product is ligated. The adapter has different base sequences including bases corresponding to polymorphic mutations, and can be labeled with different fluorescent dyes. Finally, the amplification product is labeled with a fluorescent dye corresponding to the base at the polymorphic site.

  When PCR is performed by combining a primer containing the IIs type restriction enzyme recognition sequence with a capture primer, the amplification product is fluorescently labeled and can be immobilized using the capture primer. For example, if a biotin-labeled primer is used as a capture primer, the amplification product can be captured on avidin-bound beads. By tracking the fluorescent dye of the amplification product thus captured, the base species can be determined.

[Determination of base species at polymorphic sites using magnetic fluorescent beads]
A technique capable of analyzing a plurality of alleles in parallel in a single reaction system is also known. Analyzing a plurality of alleles in parallel is called multiplexing. In general, a typing method using a fluorescent signal requires fluorescent components having different fluorescent wavelengths for multiplexing. However, there are not so many fluorescent components that can be used for actual analysis. On the other hand, when a plurality of types of fluorescent components are mixed in a resin or the like, a variety of fluorescent signals that can be distinguished from each other can be obtained even with limited types of fluorescent components. Furthermore, if a component that is magnetically adsorbed in the resin is added, it is possible to obtain beads that emit fluorescence and can be separated magnetically. Multiplex polymorphic typing using such magnetic fluorescent beads has been devised (Bioscience and Bioindustry, Vol. 60 No. 12, 821-824).

  In multiplexed polymorphic typing using magnetic fluorescent beads, a probe having a terminal complementary to the polymorphic site of each allele is immobilized on the magnetic fluorescent beads. Both are combined so that each allele corresponds to a magnetic fluorescent bead having a unique fluorescent signal. On the other hand, when the probe fixed to the magnetic fluorescent bead is hybridized to the complementary sequence, a fluorescently labeled oligo DNA having a base sequence complementary to the adjacent region on the allele is prepared.

  The allyl-containing region is amplified by asymmetric PCR, the above-described magnetic fluorescent bead-immobilized probe and a fluorescently labeled oligo DNA are hybridized, and both are further ligated. When the end of the magnetic fluorescent bead-immobilized probe has a base sequence complementary to the base of the polymorphic site, ligation is efficiently performed. On the other hand, if the terminal bases are different due to polymorphism, the ligation efficiency of the two decreases. As a result, the fluorescent labeled oligo DNA is bound to each magnetic fluorescent bead only when the sample is a base species complementary to the magnetic fluorescent bead.

  The base species is determined by collecting the magnetic fluorescent beads by magnetism and further detecting the presence of fluorescently labeled oligo DNA on each magnetic fluorescent bead. Since magnetic fluorescent beads can analyze the fluorescence signal for each bead using a flow cytometer, the signal can be easily separated even if various types of magnetic fluorescent beads are mixed. That is, “multiplexing” in which multiple types of polymorphic sites are analyzed in parallel in a single reaction vessel is achieved.

[Invader method]
A method for genotyping independent of the PCR method has also been put into practical use. For example, in the Invader method, determination of a base type is realized by only three kinds of oligonucleotides, an allele probe, an invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe requires labeling.

  The allele probe is designed to hybridize to a region adjacent to the allele to be detected. A flap consisting of a base sequence unrelated to hybridization is linked to the 5 ′ side of the allyl probe. The allyl probe has a structure that hybridizes to the 3 ′ side of the polymorphic site and is linked to a flap on the polymorphic site.

  On the other hand, the invader probe has a base sequence that hybridizes to the 5 ′ side of the polymorphic site. The base sequence of the invader probe is designed so that the 3 ′ end corresponds to the polymorphic site by hybridization. The base at the position corresponding to the polymorphic site in the invader probe may be arbitrary. That is, both base sequences are designed so that the invader probe and the allyl probe hybridize adjacently across the polymorphic site.

  If the polymorphic site is complementary to the base sequence of the allele probe, both the invader probe and the allele probe hybridize to allele, and the invader probe enters the base corresponding to the allele probe polymorphic site. An (invasion) structure is formed. The cleavase cleaves the invading strand of the oligonucleotide that has formed the invading structure thus formed. Since the cleavage occurs on the intrusion structure, the result is that the allyl probe flap is severed. On the other hand, if the base at the polymorphic site is not complementary to the base of the allyl probe, there is no competition between the invader probe and the allyl probe at the polymorphic site, and no invasion structure is formed. Therefore, the flap is not cut by cleavase.

  The FRET probe is a probe for detecting the flap thus separated. The FRET probe constitutes a hairpin loop having a self-complementary sequence on the 5 ′ end side and a single-stranded portion arranged on the 3 ′ end side. The single-stranded portion arranged on the 3 ′ end side of the FRET probe has a base sequence complementary to the flap, and the flap can hybridize there. Both base sequences are designed so that when the flap hybridizes to the FRET probe, a structure is formed in which the 3 ′ end of the flap enters the 5 ′ end of the self-complementary sequence of the FRET probe. A cleavase recognizes and cleaves an invasion structure. If the FRET probe is cleaved by a cleavase and labeled with a reporter dye and quencher similar to TaqMan PCR, the FRET probe cleavage can be detected as a change in fluorescence signal.

  Theoretically, the flap should hybridize to the FRET probe even in the uncut state. However, in reality, there is a large difference in the binding efficiency to FRET between the cleaved flap and the flap present in the form of an allyl probe. Therefore, it is possible to specifically detect the cleaved flap using the FRET probe.

  In order to determine the base species based on the Invader method, two types of allyl probes including base sequences complementary to allyl A and allyl B may be prepared. At this time, the base sequences of the flaps are different base sequences. If two types of FRET probes for detecting flaps are prepared and each reporter dye can be identified, the base species can be determined based on the same concept as the TaqMan PCR method.

  The advantage of the Invader method is that the FRET probe is the only oligonucleotide that needs to be labeled. The FRET probe can use the same oligonucleotide regardless of the base sequence to be detected. Therefore, mass production is possible. On the other hand, since the allyl probe and the invader probe do not need to be labeled, a reagent for genotyping can be manufactured at low cost.

[RCA method]
The RCA method can be mentioned as a method for determining the base species independent of the PCR method. A method for amplifying DNA based on a reaction in which a DNA polymerase having a strand displacement action synthesizes a long complementary strand using a circular single-stranded DNA as a template is the Rolling Circle Amplification (RCA) method (Lizardri PM et al., Nature Genetics 19, 225, 1998). In the RCA method, an amplification reaction is configured using a primer that anneals to a circular DNA and initiates complementary strand synthesis and a second primer that anneals to a long complementary strand generated by this primer.

  In the RCA method, a DNA polymerase having a strand displacement action is used. Therefore, the portion that has become double-stranded by complementary strand synthesis is replaced by a complementary strand synthesis reaction started from another primer annealed to the 5 ′ side. For example, the complementary strand synthesis reaction using circular DNA as a template does not end in one round. The complementary strand synthesis continues while replacing the previously synthesized complementary strand, and a long single-stranded DNA is produced. On the other hand, the second primer anneals to the long single-stranded DNA generated using the circular DNA as a template, and complementary strand synthesis starts. Since single-stranded DNA generated by the RCA method uses circular DNA as a template, its base sequence is a repetition of the same base sequence. Thus, continuous production of long single strands results in continuous annealing of the second primer. As a result, single-stranded portions that can be annealed by the primer are continuously generated without going through a denaturing step. Thus, DNA amplification is achieved.

  If the circular single-stranded DNA necessary for the RCA method is generated according to the base type of the polymorphic site, the base type can be determined using the RCA method. For this purpose, a linear single-stranded padlock probe is used. The padlock probe has complementary base sequences on both sides of the polymorphic site to be detected at the 5 ′ end and 3 ′ end. These base sequences are linked at a portion consisting of a special base sequence called a backbone. If the polymorphic site is a base sequence complementary to the end of the padlock probe, the end of the padlock probe hybridized to the allele can be ligated by DNA ligase. As a result, the linear padlock probe is circularized and the reaction of the RCA method is triggered. The reaction of DNA ligase is significantly reduced in efficiency when the terminal portion to be ligated is not completely complementary. Therefore, the base type of the polymorphic site can be determined by confirming the presence or absence of ligation by the RCA method.

  The RCA method can amplify DNA but does not generate a signal as it is. Moreover, if only the presence or absence of amplification is used as an index, the base species cannot usually be determined unless the reaction is performed for each allele. Methods are known in which these points are improved for the determination of base species. For example, using a molecular beacon, the base type can be determined in one tube based on the RCA method. A molecular beacon is a signal generation probe using a fluorescent dye and a quencher, as in the TaqMan method. The molecular beacons are composed of complementary base sequences at the 5 ′ end and 3 ′ end, and by themselves form a hairpin structure. If both ends are labeled with a fluorescent dye and a quencher, a fluorescent signal cannot be detected in a state where a hairpin structure is formed. If a part of the molecular beacon is set as a base sequence complementary to the amplification product of the RCA method, the molecular beacon hybridizes to the amplification product of the RCA method. Since the hairpin structure is eliminated by hybridization, a fluorescent signal is generated.

  The advantage of the molecular beacon is that the base sequence of the molecular beacon can be made common regardless of the detection target by using the base sequence of the backbone portion of the padlock probe. If the base sequence of the backbone is changed for each allele and two types of molecular beacons having different fluorescence wavelengths are combined, the base type can be determined in one tube. Since the cost of synthesis of fluorescently labeled probes is high, the ability to use a common probe regardless of the measurement target is an economic advantage.

  All of these methods have been developed for genotyping a large amount of samples at high speed. With the exception of MALDI-TOF / MS, it is usually necessary to prepare a labeled probe or the like in any way for either method. On the other hand, a method for determining a base type that does not rely on a labeled probe has been performed for a long time. As one of such methods, for example, a method using restriction fragment length polymorphism (RFLP), a PCR-RFLP method and the like can be mentioned.

  RFLP utilizes the fact that a restriction enzyme recognition site mutation or a base insertion or deletion in a DNA fragment caused by restriction enzyme treatment can be detected as a change in the size of the fragment produced after the restriction enzyme treatment. If there is a restriction enzyme that recognizes the base sequence containing the polymorphism to be detected, the base of the polymorphic site can be known by the principle of RFLP.

  As a method that does not require a labeled probe, a method for detecting a difference in base using a change in the secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its nucleotide sequence (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). The PCR-SSCP method is performed by dissociating the PCR product into single-stranded DNA and separating it on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.

  Other examples of methods that do not require a labeled probe include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated depending on the instability of each. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of a partially dissociated DNA fragment becomes very slow and can be separated from the mobility of a complete double-stranded DNA without a dissociated portion.

  Specifically, first, a region containing a polymorphic site is amplified by PCR or the like. The amplification product is hybridized with a probe DNA whose base sequence is known to make a double strand. This is electrophoresed in a polyacrylamide gel that gradually increases as the concentration of denaturing agents such as urea moves, and is compared with a control. In a DNA fragment that has mismatched by hybridization with the probe DNA, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the moving speed becomes extremely slow. The presence or absence of mismatch can be detected by detecting the difference in mobility generated in this way.

  In addition, the DNA species can be used to determine the base species (Cell engineering separate volume “DNA microarray and the latest PCR method”, Shujunsha, published 2000.4 / 20, pp97-103 “SNP analysis using oligo DNA chip”, Sabae. Shinichi). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously.

  In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous membrane such as a nitrocellulose membrane can also be used.

  In the present invention, examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Affymetrix. In an oligonucleotide array, oligonucleotides are usually synthesized in vitro. For example, in-situ synthesis methods of oligonucleotides using photolithographic technology (Affymetrix) and inkjet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known. Can be used.

  The oligonucleotide is composed of a base sequence complementary to a region containing the SNPs to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases when the oligonucleotide is immobilized. Furthermore, in general, mismatch (MM) probes are used in the DNA array method in order to avoid errors due to cross hybridization (non-specific hybridization). The mismatch probe constitutes a pair with an oligonucleotide having a base sequence completely complementary to the target base sequence. An oligonucleotide consisting of a base sequence that is completely complementary to a mismatch probe is called a perfect match (PM) probe. In the process of data analysis, the influence of cross-hybridization can be reduced by eliminating the signal observed with the mismatch probe.

  A sample for genotyping by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from chromosomal DNA extracted from a subject's blood, tissue or cells such as peripheral blood leukocytes, skin, oral mucosa, tears, saliva, urine, feces or hair. A specific region of chromosomal DNA is amplified using a primer for amplifying a region containing a polymorphic site to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing multiple polymorphic sites, the multiplex PCR method is useful.

  In general, in the DNA array method, a DNA sample is amplified by a PCR method and an amplification product is labeled. A labeled primer is used for labeling the amplification product. For example, genomic DNA is first amplified by PCR using a primer set specific to the region containing the polymorphic site. Next, biotin-labeled DNA is synthesized by a labeling PCR method using a biotin-labeled primer. The biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the substrate and the reaction temperature. One skilled in the art can design appropriate hybridization conditions. In order to detect the hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.

  More specifically, after obtaining the DNA comprising the polymorphic site of the present invention prepared from the subject and the solid phase on which the nucleotide probe is immobilized, the DNA and the solid phase are then obtained. Make contact. Furthermore, the base type of the polymorphic site of the present invention is determined by detecting DNA hybridized to the nucleotide probe immobilized on the solid phase.

  In the present invention, “solid phase” means a material capable of fixing nucleotides. The solid phase of the present invention is not particularly limited as long as nucleotides can be immobilized. Specifically, solid phases including microplate wells, plastic beads, magnetic particles, substrates and the like may be exemplified. it can. As the “solid phase” of the present invention, a substrate generally used in DNA array technology can be preferably used. In the present invention, the “substrate” means a plate-like material capable of fixing nucleotides. In the present invention, the nucleotide includes oligonucleotides and polynucleotides.

In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allyl-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a polymorphic site to be detected is present. When ASO is hybridized to sample DNA, the hybridization efficiency decreases if a mismatch occurs at the polymorphic site due to the polymorphism. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.
Among the above oligonucleotides, the CCL1 gene or a site on the DNA region in the vicinity of the gene, which hybridizes to DNA containing the polymorphic site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 1, An oligonucleotide having a chain length can be used as a reagent (test agent) for testing whether COPD is susceptible to infection. This is used for a test using gene expression as an index or a test using gene polymorphism as an index.

  The oligonucleotide specifically hybridizes to DNA containing the polymorphic site of the CCL1 gene of the present invention. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not need to be completely complementary to the nucleotide sequence in the gene or in the DNA region adjacent to the gene.

  The oligonucleotide can be used as a probe or primer in the test method of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of DNA containing any polymorphic site of the CCL1 gene of the present invention.

  The present invention provides a primer for amplifying a region containing the polymorphic site of the present invention, and a probe that hybridizes to a DNA region containing the polymorphic site.

  In the present invention, the primer for amplifying a region containing a polymorphic site also includes a primer that can initiate complementary strand synthesis toward the polymorphic site using DNA containing the polymorphic site as a template. The primer can also be expressed as a primer for providing a replication origin on the 3 ′ side of the polymorphic site in DNA containing the polymorphic site. The interval between the region where the primer hybridizes and the polymorphic site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the polymorphic site. For example, in the case of a primer for analysis using a DNA chip, the primer can be designed so as to obtain an amplification product having a length of 20 to 500, usually 50 to 200 bases, as a region containing a polymorphic site. A person skilled in the art can design a primer according to the analysis method based on the base sequence information about the surrounding DNA region including the polymorphic site. The base sequence constituting the primer of the present invention can be appropriately modified as well as a base sequence completely complementary to the genomic base sequence.

  In addition to the base sequence complementary to the genomic base sequence, an arbitrary base sequence can be added to the primer of the present invention. For example, in a primer for a polymorphism analysis method using a type IIs restriction enzyme, a primer to which a recognition sequence for a type IIs restriction enzyme is added is used. Such a primer with a modified base sequence is included in the primer of the present invention. Furthermore, the primer of the present invention can be modified. For example, a fluorescent substance or a primer labeled with a binding affinity substance such as biotin or digoxin is used in various genotyping methods. Primers having these modifications are also included in the present invention.

  On the other hand, in the present invention, a probe that hybridizes to a region containing a polymorphic site refers to a probe that can hybridize to a polynucleotide having a base sequence of a region containing a polymorphic site. More specifically, a probe containing a polymorphic site in the base sequence of the probe is preferable as the probe of the present invention. Alternatively, depending on the base analysis method at the polymorphic site, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.

  In other words, a probe capable of hybridizing to the polymorphic site of the present invention on genomic DNA or a site adjacent to the polymorphic site is preferred as the probe of the present invention. In the probe of the present invention, modification of the base sequence, addition of the base sequence, or modification is allowed in the same manner as the primer. For example, a probe used for the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of the present invention in the genome.

  The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the base sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, usually 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  The present invention also provides a reagent (test agent) for use in the test method of whether or not the COPD of the present invention is susceptible to infection (has a predisposition to susceptibility). The reagent of the present invention includes the primer and / or probe of the present invention. In the examination of whether or not an infectious disease is susceptible in COPD, a DNA containing a CCL1 gene or a site on the DNA region in the vicinity of the CCL1 gene and containing a polymorphic site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 1. Primers and / or probes are used for amplification.

  Various reagents, enzyme substrates, buffers and the like can be combined with the reagent of the present invention depending on the method for determining the base species. As the enzyme, enzymes necessary for various analysis methods exemplified as the above-mentioned base species determination method such as DNA polymerase, DNA ligase, or IIs restriction enzyme can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

  Furthermore, the reagent of the present invention can be accompanied by a control in which the base at the polymorphic site is clear. As a control, a genome or a genomic fragment in which the base type of the polymorphic site is known in advance can be used. The genome may be extracted from cells, or cells or cell fractions may be used. If a cell is used as a control, the result of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA comprising a base sequence containing a polymorphic site can be used as a control. Specifically, a YAC vector or a BAC vector containing a genome-derived DNA whose base species at the polymorphic site of the present invention has been clarified is useful as a control. Alternatively, a vector in which only several hundred bases corresponding to the polymorphic site are cut out and inserted can be used as a control.

Furthermore, another aspect of the reagent of the present invention is to examine whether or not AECOPD is susceptible, comprising a solid phase to which a nucleotide probe that hybridizes with DNA containing the polymorphic site of the CCL1 gene of the present invention is immobilized. It is a reagent.
These are used for examinations using the polymorphic site of the present invention as an index. These preparation methods are as described above.

  In addition, the present invention uses the expression level of the CCL1 gene in a subject (biological sample derived from the subject) as an index, whether or not it is susceptible to AECOPD, or when COPD develops in the future without COPD It is also possible to determine whether or not there is a predisposition to AECOPD susceptibility. That is, according to the present invention, when the expression level of the CCL1 gene in the subject is reduced as compared with the control, the subject is determined to be susceptible to AECOPD or predisposed to AECOPD in COPD. A method for testing whether or not AECOPD is susceptible or predisposed to AECOPD susceptibility is provided.

  In the above method, a biological sample derived from a subject is usually used as a test sample. Measurement of the expression level of the CCL1 gene in the test sample can be appropriately performed by those skilled in the art using known techniques. The above “control” usually refers to the expression level of the CCL1 gene in a biological sample derived from a healthy person. The expression of the CCL1 gene in the present invention means both the expression of mRNA transcribed from the CCL1 gene and the expression of the protein encoded by the CCL1 gene.

  Furthermore, the present invention provides a screening method for a therapeutic or prophylactic agent for AECOPD. The inventors have confirmed through experiments that the risk allele at the SNP rs2282691 site, which has been found to be associated with AECOPD, is less likely to bind C / EBPβ, which is a gene transcription enhancer, than the protective allele. Therefore, suppression of transcription of the CCL1 gene is thought to increase the risk of developing AECOPD, and if a substance that activates the expression level of the CCL1 gene or the function of the protein encoded by the CCL1 gene is selected, that is, it is a therapeutic agent for AECOPD. Or it is expected to become a prophylactic agent.

The above-mentioned method of the present invention is a screening method for selecting a therapeutic or prophylactic agent for AECOPD, including, for example, the following steps (a) to (c).
(A) a step of bringing a test substance into contact with a cell expressing the CCL1 gene; (b) a step of measuring the expression level of the CCL1 gene; and (c) the expression compared to a case where the test substance is measured in the absence of the test substance. The process of selecting substances that increase the level

  In this method, a test substance is brought into contact with cells expressing the CCL1 gene. As the cells to be used, cells derived from humans, animals, insects and the like can be used. A cell having an endogenous CCL1 gene can be used, or a CCL1 gene can be further introduced into a cell having an endogenous CCL1 gene. In addition, an exogenous CCL1 gene can be introduced into cells lacking the CCL1 gene and used in experiments. Gene transfer can be performed by a well-known general method using a vector.

  There is no particular limitation on the test substance used in this method. A low molecular compound, a high molecular compound, a natural product, a protein, a peptide, or the like, or a cell extract, a cell culture supernatant, or the like can be used, but is not limited thereto.

  In this method, the test substance can be brought into contact with the cell expressing the CCL1 gene by adding the test substance to the culture solution of the cell expressing the CCL1 gene, but is not limited to this method.

The expression level of the CCL1 gene can be measured by a generally known measurement technique such as RT-PCR. Further, the expressed CCL1 protein can be measured by a generally known measurement method such as ELIZA method or Western blot method.
Next, a test substance that increases the expression level of the CCL1 gene is selected in the absence of the test substance, that is, when the test substance is not contacted with cells. The selected test substance becomes an AECOPD therapeutic agent or prophylactic agent.

As another aspect of the screening method of the present invention, a reporter gene assay for selecting a substance that increases the expression level of CCL1 including the following steps (a) to (c) can be mentioned.
(A) a step of bringing a test substance into contact with a cell or a cell extract containing DNA having a structure in which a transcriptional regulatory region of the CCL1 gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene Step (c) A step of selecting a substance that increases the expression level as compared with the case where it is measured in the absence of the test substance.

  In this method, a test substance is brought into contact with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of the CCL1 gene and a reporter gene are functionally linked. Those skilled in the art can obtain gene sequence information of the transcriptional regulatory region of the CCL1 gene from various databases including Genbank. The reporter gene expression is induced by binding a transcription factor to the transcriptional regulatory region using a generally known reporter gene assay technique, the reporter gene expression level is measured, and the test substance is not contacted. It is possible to select a test substance that promotes the transcription of the CCL1 gene by comparing the expression level of the reporter gene with the case as a control. A test substance that promotes transcription of the CCL1 gene can be used as a therapeutic or prophylactic agent for AECOPD.

As still another aspect of the present invention, a screening method using the binding activity between a polynucleotide containing a C / EBPβ recognition sequence and C / EBPβ protein as an index, including the following steps (a) to (c): be able to.
(A) contacting a polynucleotide containing a C / EBPβ recognition sequence and C / EBPβ protein with a test substance (b) measuring a binding activity between the polynucleotide and a transcription factor (c) absence of the test substance A step of selecting a substance that enhances the binding activity as compared to the case measured below

  In this method, the polynucleotide containing the C / EBPβ recognition sequence is brought into contact with the C / EBPβ protein and the test substance, and the test substance promotes the binding between the polynucleotide containing the C / EBPβ recognition sequence and the C / EBPβ protein. You can consider whether or not. For example, although not limited to the method described below, incubation is performed by adding an anti-C / EBPβ protein antibody to a C / EBPβ protein and a polynucleotide containing a C / EBPβ recognition sequence by EMSA, which is a generally known method. Test substance that changes the binding activity between the polynucleotide containing the C / EBPβ recognition sequence and the C / EBPβ protein by adding the test substance and comparing the position and concentration of the band that appears in the absence of the test substance Can be selected. A test substance that increases the binding activity between a polynucleotide containing a C / EBPβ recognition sequence and C / EBPβ protein can be used as a therapeutic or prophylactic agent for AECOPD.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

<Research Group>
Among Japanese COPD patients who visited an outpatient clinic at Yamagata University Hospital (Japan) in 2001-2005, 276 male patients whose genomic DNA was available after informed consent on genotyping was obtained in writing We investigated people. These COPD patients were diagnosed according to criteria established by the American Thoracic Society (American Thoracic Society. Am J Respir Crit Care Med. 1995; 152: S77-S120.). Irreversible chronic airflow obstruction was confirmed by spirogram for all patients. Patients with complications such as chronic heart failure, autoimmune disease, malignancy were excluded. None of the patients were on nutritional supplements. All target patients receive influenza vaccine every winter. All patients were anthropometrically measured before breakfast on the day of the examination. The study protocol was approved by the Yamagata University School of Medicine Ethics Committee and written informed consent was obtained from all patients prior to participating in the study.

<Retrospective and prospective cohort study>
Prior to the prospective study, retrospective data on enrolled COPD patients were collected from January 1, 2001 to December 31, 2002. As of January 1, 2003, AECOPD frequency was obtained from blood samples from a total of 276 male COPD patients and their respective medical records. Two separate pulmonologists carefully assessed each patient's medical records for the past two years, and only those cases where both pulmonologists were both AECOPD were counted as AECOPD. Diagnosis of AECOPD, as is commonly done, in addition to increased sputum volume, color change, viscosity change, increased dyspnea, increased cough, fever, whole body A comprehensive judgment was made on the deterioration of the condition. The inventors divided the incidence and severity of AECOPD into a retrospective cohort study and a prospective cohort study, respectively. This improved the accuracy of the study.
Through retrospective and prospective cohort studies, patients were followed up on an outpatient basis every month, as well as when AECOPD was suspected. A thorough clinical evaluation was performed by a general physician at each regular visit. When the patient was suspected of AECOPD, one of the two respiratory physicians participated in the clinical evaluation. Although the diagnostic criteria for AECOPD have not yet been established (MacNee W. Acute exacerbations of COPD. Swiss Med Wkly. 2003; 133: 247-257.), The inventors performed the diagnosis as follows. Subjects are first asked six questions regarding overall health, dyspnea, cough, sputum volume, sputum consistency, and whether sputum is purulent. Three major symptoms were weighted. That is, the amount of sputum, whether the sputum is purulent (determined by color tone), and dyspnea. The subjects are then evaluated for overall clinical findings, respiratory rate, wheezing and rales. Based on these observations, the inventors determined whether the patient was in a clinically stable state or in an acute exacerbation (Abe Y et al., Am J Respir Crit Care Med. 2002; 165: 967-971., Bakri F et al., J Infect Dis. 2002; 185: 632-640.).
Using the clinical information obtained from these retrospective studies, we analyzed the association between the typed SNP and the frequency of AECOPD. In addition, the inventors continued to look forward to the same patient group (276 male COPD patients) for 30 months from January 1, 2003 to June 30, 2005. A prospective study evaluated the severity of typed SNPs and AECOPD (endpoint: death from AECOPD). Furthermore, the fact that it was severe AECOPD was proved by quantification of serum C-reactive protein (CRP), or an increase in the number of white blood cells in the blood, pneumonia, etc. by findings by X-ray photography.

<Lung function test>
Forced vital capacity (FVC) and 1-second volume (FEV1.0) were measured with a vital capacity measuring instrument (CHESTAC-25 Part II EX; Chest Co., Ltd., Tokyo, Japan) by a standard method. At least 3 spirometry was performed and the highest value was used. The reference value was proposed by the Japanese Society of Chest Diseases (Japanese Society of Chest Disease. Jpn J Thorac Dis 1993; 31: appendix.). Arterial blood gas analysis was performed on subjects breathing with or without supplemental oxygen in the sitting position (280 Blood Gas System; Ciba Corning Diagnostics Corp., Medfield, Mass.).

<Candidate SNP selection and polymorphic genotyping>
94 genes that may be related to COPD, which are known to be mainly involved in immunity and inflammation, were selected, and SNPs on each gene were selected as dbSNP (http://www.ncbi.nlm.nih.gov/ SNP /), JSNP (http://snp.ims.u-tokyo.ac.jp), and Applied Biosisytems genotyping Database (http://myscience.appliedbiosystems.com/ extracted from genotype /). 200 SNPs were selected, and all SNPs were analyzed for the frequency of AECOPD (more than 2 times / 2 years vs. no onset for 2 years) and the association with AECOPD death.
In addition, in order to search for candidate transcription factors that bind to surrounding sequences containing the selected SNP, Match (Ver.1.4.1), a computer program sold by the TRANSFAC Professional database (Biobase Biological Database), was used to score. Transcription factors were searched with a threshold of 85 or more (Table 2b, 2c) (Kochi Y et al., Nat Genet. 2005; 37: 478-485., Wang J et al., Mol Cell Biol. 2004; 24: 2423 -4243., Davis ME et al., J Biol Chem. 2004; 279: 163-168., Tullai JW et al., J Biol Chem. 2004; 279: 20167- 20177.). In a prospective cohort study, CCL1, CCL5, and CCL11 were correlated with the incidence of AECOPD in the vicinity, so CCL1 and its nearby chemokines were also included in the SNP analysis. The positions of CCL1, CCL11 and CCL5 on the chromosome are shown in FIG. 2 SNPs of CCL11, 1 SNP of CCL1, and 1 SNP of CCL5 were typed. In a prospective cohort study, the same COPD patient population was used to analyze the severity of these SNPs and AECOPD.

<SNP genotyping method>
Genetic polymorphism SNP genotyping analysis was performed by TaqMan allelic discrimination assay (Livak KJ. Genet Anal. 1999; 14: 143-149.). Reagents were purchased from Applied Biosystems (Foster City, CA, USA). A Taqman probe for distinguishing SNPs upon completion of the PCR reaction was designed and synthesized by Applied Biosisytems. One allele probe was labeled with a fluorescent FAM dye and the other was labeled with a fluorescent VIC dye. PCR reactions were performed in TaqMan Universal Master Mix without UNG (Applied Biosisytems) without UNG, using PCR primers at a concentration of 900 nM and Tackman MGB-probe at a concentration of 200 nM. The reaction was performed in a 384-well format using 3.0 ng genomic DNA with a total reaction volume of 3 μl. Next, the plate was placed in GeneAmp PCR System 9700 (Applied Biosisytems), heated to 95 ° C. for 10 minutes, then subjected to a cycle of 92 ° C. for 15 seconds and 60 ° C. for 1 minute, and finally immersed in 25 ° C. The fluorescence intensity in each well of the plate was read with a Prism 7900HT instrument (Applied Biosisytems). Fluorescence data files from each plate were analyzed with SDS 2.0 allele calling software (Applied Biosisytems).

<Statistical analysis>
The chi-square test tested whether each allele matched Hardy-Weinberg equilibrium (HWE) (p> 0.05). Genotype and susceptibility to AECOPD (incidence of infection (more than 2/2 years vs. 0 times / 2 years), death from AECOPD (from a 30-month prospective study)) and other clinical parameters Was evaluated by Fisher's direct method, logistic regression analysis. Both dominant and recessive genetic models were applied to these statistical analyses. Because age, lung function index, and smoking index are potential confounding variables in COPD patients and multivariate analysis needs to compensate for the effects of these confounding variables, logistic regression analysis uses age, smoking Adjustments were made for index, body mass index (BMI) and lung function index (forced expiratory volume per second-percentage of expected value). In the analysis for all target COPD patients, the Kruskal-Wallis test (non-parametric ANOVA) was used to evaluate the difference in the average number of AECOPD frequency ranks in different genotype groups. Survival analysis from AECOPD was a prospective cohort study that analyzed the 30-month follow-up period using univariate analysis using the Kaplan-Meier method and the log-rank test. The survival analysis from AECOPD was also performed using Cox's proportional hazards model adjusted for age, BMI, and lung function index as covariates that could affect overall mortality (Schols). AM, et al., Am J Respir Crit Care Med. 1998; 157: 1791-1797). Results were expressed as mean ± standard deviation (SD), and P <0.05 was considered significant. All data analysis is SPSS ver.12.0.1J (SPSS Inc., Chicago, IL, 2003). It was performed using.

<Nuclear extract and gel shift assay (EMSA: Elecgtromobility Shift Assay)>
A monocyte-derived cultured cell line, THP-1, was cultured in PRIMI1640 (GIBCO) supplemented with 10% FBS (fetal bovine serum). After adjusting the cell concentration to 1.0 × 106 / ml, medium containing human recombinant interleukin-6 (IL-6) (10 ng / ml) and human recombinant gamma interferon (IFN-γ) (400 units / ml) And the cell concentration was adjusted to 1.0 × 10 6 / ml. After 6 hours of culture stimulation, cells were collected and a nuclear extract was prepared as previously reported (Wong HR, et al., J Clin Invest. 1997; 99: 2423-2428). The oligonucleotide used for EMSA was designed using TRANSFAC professional database (Biobase). CCL1-SNP3- [CCAAT / enhancer binding protein beta (C / EBP?)] -A Allele-sense (5'-ATTGGAGTAGCTTTCACAAACCAGTCAGTAT-3 ') sequence is shown in Sequence Listing 2 and CCL1-SNP3-C / EBP? -A The sequence of allyl-antisense (5'-ATACTGACTGGTTTGTGAAAGCTACTCCAAT? 3 ') is shown in Sequence Listing 3, and the sequence of CCL1-SNP3-C / EBP? -B allele-sense (5'-ATTGGAGTAGCTTTCACATACCAGTCAGTAT? 3') is shown in Sequence Listing 4. The sequence of CCL1-SNP3-C / EBP? -B allyl-antisense (5'-ATACTGACTGGTATGTGAAAGCTACTCCAAT? 3 ') is shown in Sequence Listing 5.
Nucleoprotein (5 μg) was mixed with the labeled probe and incubated for 20 minutes at room temperature. The mixture was electrophoresed on a polyacrylamide gel and transferred to a nylon membrane. Band detection was performed using the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology) according to the instruction manual. In addition, the specificity of the band was confirmed by a competition experiment in which an unlabeled probe with a consensus sequence for C / EBPβ was added in excess before mixing the nuclear extract and the labeled probe. Furthermore, a supershift assay for C / EBPβ protein was performed using an anti-C / EBPβ antibody (Santa Cruz Biotechnology). Nuclear extracts and anti-C / EBPβ antibody (4 μg) were incubated for 1 hour at 4 ° C. before adding labeled probe.

[Example 1] Correlation analysis of a retrospective study First, patients who developed AECOPD more than once in the past 2 years (assumed to be group C) (n = 73, AECOPD frequency: 4.27 ± 5.41 times (range: 2-40) Times)) and patients who did not develop obvious AECOPD (group c) (n = 123) (Table 3).
Patients in group C had significantly lower BMI (kg / m ² ) (19.9 ± 3.3 vs. 20.9 ± 3.2, p <0.05) and% FEV1.0 (% predicted) (43.4 ± 22.6 vs. c) 49.8 ± 20.4, p <0.01). The SNP (rs2282691) on the CCL1 gene (Miller MD et al., Proc Natl Acad Sci USA, 1992; 89: 2950-2954), which encodes a monocyte-specific chemoattractant, is the Fisher's direct test (Odds ratio (ORs ): 2.70, 95% confidence interval (95% CI): 1.36-5.36) p = 0.004) and logistic regression analysis (odds ratio (OR): 3.06, 95% confidence interval (95% CI): 1.46-6.41) p = 0.003) and a significant association with the frequency of AECOPD was found in the dominant model test (Table 2a).
The frequency of hate was a worsening of% FEV1.0 (predicted%) representing disease severity (Burge PS et al., BMJ. 2000; 320: 1297-1303) and BMI predicting a poor clinical prognosis Decreased (Takabatake N et al., Am J Respir Crit Care Med. 2001; 163: 1314-1319., Takabatake N et al., Am J Respir Crit Care Med. 2000; 161: 1179-1184. Takabatake N et al. , Am J Respir Crit Care Med. 1999; 159: 1215-1219.), A multivariate analysis was performed to exclude the possibility that low lung function index and low BMI worked as confounding factors. As shown in Table 4b, SNP rs2282691 on the CCL1 gene showed a significant difference in the dominant model (AA + AT vs. TT) considering the difference in prognostic factors between group C and group c (odds ratio ( OR): 2.82, 95% confidence interval (95% CI): 1.39-5.71), p = 0.004). Conversely, BMI and% FEV1.0 (% predicted) were not significant risk factors for AECOPD.
The CCL subfamily of chemokine genes is clustered in the chromosomal region 17q11.2-q12, but CCL11 (SNP rs17809012, rs4795900) and CCL5 (SNP rs3817655) present in the vicinity of the CCL1 gene No association with susceptibility to acute respiratory tract infection (acute exacerbation) (Table 2)

[Example 2] Correlation analysis of a retrospective cohort study The Kruskal-Wallis test (non-parametric) was used to compare the difference in the average value of the frequency of AECOPD in each of the three genotypes according to the incidence of AECOPD in a retrospective study. ANOVA) was applied. In this analysis, four patients who did not achieve an accurate AECOPD incidence were excluded from the analysis. SNP rs2282691 on CCL1 showed a significant p-value (p = 0.006, Table 5a), and the risk allele was 'A' (p = 0.003, Table 5b) assuming a dominant model. In addition, when a recessive model was assumed, no significant correlation was observed (Table 5c), and therefore the number of AECOPD was higher (Tables 5a and 5b). These results were consistent with Fisher's direct test results and logistic regression analysis results. The total ANP frequency of SNP rs2282691 for all subjects was 0.456, the T allele frequency was 0.544, and the Hardy-Weinberg equilibrium was 0.365 (p = 0.546 (chi-square test)).

[Example 3] Kaplan-Meier analysis using log rank test of prospective cohort study The purpose of the prospective study was to evaluate the association between the genotype of SNP rs2282691 on CCL1 and the severity (endpoint: death) of AECOPD is there. None of the patients dropped out or were excluded except when they died. The Kaplan-Meier method was applied according to the results of the retrospective cohort study (Table 5), and whether or not there was a difference in the tendency of the survival curve for genotypes and alleles was evaluated using a log rank test. This analysis is based on a follow-up study conducted over 30 months (Table 6).

  As a result of analyzing the follow-up data for 30 months by the Kaplan-Meier method, it was found that the 'A' allele was a risk allele of significant AECOPD severity. This result indicates that A 'allele is a risk allele of AECOPD death in this prospective study. Having an 'A' allele shortens the average survival to death from AECOPD. Further, it was found that genotype AA having a homonymous 'A' allyl has a stronger and stronger correlation than genotype AT which is hetero. Genotype AT that is heterozygous did not show a significant correlation with TT genotype that is homozygous for 'T' allele (P = 0.06), but log rank analysis showed significant differences in both dominant and recessive models (Recessive model: log rank analysis 4.43, p = 0.0353). The reason for this result is that for the severity (mortality) of AECOPD, the heterogenotype is also considered a risk factor, and the typical gene dosage effect of the negative impact of risk allele 'A' on survival is Yes (Figure 2).

Example 4 Cox Proportional Hazard Regression Analysis in a Prospective Cohort Study The inventors next analyzed the 30-month follow-up data using the Cox proportional hazard regression analysis method. The Cox proportional hazard regression analysis model can be effectively applied to the results of the inventors, and a proportional hazard tendency was observed in SNP rs2282691 on the CCL1 gene (Table 7). In both genotype and 'A' allyl dominant models, age was recognized as a risk factor for the severity (mortality) of AECOPD (p = 0.041 and p = 0.038, respectively). Early in the prospective study, nutritional status (BMI) and lung function index (% FEV1.0 (% of expected value)) were not significant, but these factors contributed to the severity (mortality) of AECOPD The results of the analysis are shown in Fig. 3. Fig. 3 was similar to the results of Kaplan-Meier analysis in this analysis, and the typical gene dosage effect was also the same. Recognized.

  In this prospective study, a total of 47 patients died. Of the 47, 23 patients died of AECOPD (48.9%). As a result of conducting a culture test of these 23 dead people, pathogenic bacteria having a possibility of pathogenicity were found in the sputum of 12 patients. Breaking down, methicillin-resistant Staphylococcus aureus (MRSA) was identified as a single pathogen in five patients. Methicillin-sensitive Staphylococcus aureus (MSSA) has been identified as a single pathogen in two patients. MRSA and Pseudomonas aeruginosa (1), MRSA and Klebsiella pneumoniae (1), MRSA and penicillin-sensitive Strepcoccus pneumoniae (PSPP) (1), Pseudomonas aeruginosa (1) One was Klebsiella pneumoniae, one was MSSA, Klebsiella pneumoniae and one Haemophilus parainfluenzae, and the remaining 11 sputum specimens showed non-specific culture findings.

[Example 5] EMSA
To analyze the function of SNP rs2282691 present in intron 2 of the CCL1 gene, the TRANSFAC Professiona database (Kochi Y et al., Nat Genet. 2005; 37: 478-485., Wang J, et al., Mol Cell Biol 2004; 24: 2423-4243., Davis ME, et al., J Biol Chem. 2004; 279: 163-168., Tullai JW, et al., J Biol Chem. 2004; 279: 20167- 20177.) Was used to search for transcription factors that could bind to this part. The inventors have found candidates for transcription factors that do not bind to the 'A' allyl but bind to the 'T' allyl. Two of C / EBPβ and transcription factor poly (c) -binding protein 2 (TFCP2) had TRANSFAC scores of 89 and 87, respectively (Tables 2b and 2c). Therefore, the inventors performed EMSA to examine whether the sequence around SNP rs2282691 binds to C / EBPβ. A nuclear extract of THP-1, a cultured cell of mononuclear cells, was prepared after stimulation with XX, and was prepared using part of the CCL1 gene sequence with SNP rs2282691 part 'A' or 'T' EMSA was performed using oligonucleotides. As for the complex of the nuclear extract and the oligonucleotide, DNA-nucleoprotein complex was observed in the gel electrophoresis with the 'T' oligonucleotide (Fig. 4, lane 2), but with the DNA-nucleoprotein complex with the 'A' oligonucleotide. No body was observed (FIG. 4, lane 5). Specificity was confirmed by a competitive reaction with unlabeled DNA (FIG. 4, lane 4). Please re-enter after confirming the explanation of the figure. In addition, this complex was supershifted by the addition of anti-C / EBPβ antibody (FIG. 4, lane 3). These results indicate that the 'T' allyl of SNP rs2282691 binds to C / EBPβ, but the 'A' allyl does not bind to C / EBPβ.

Other chemokines targeted for SNP analysis in the vicinity of the CCL1 gene on the chromosome. CCL11, CCL1, and CCL5 form a cluster on chromosome 17. Based on the results of a 30-month prospective cohort study, survival curves were drawn by the Kaplan-Meier method with the genotype of SNP rs2282691 on the CCL1 gene and the 'A' allele dominant model, and the trend was analyzed using the log rank test. + In the figure indicates examples of censoring due to causes other than AECOPD. See Table 6 for statistical analysis results. (A) Three survival curves were drawn for each genotype. (B) Two survival curves drawn assuming a dominant model of 'A' allyl. Survival curves from Cox proportional hazard regression model with SNP rs2282691 genotype and 'A' allele dominant model on CCL1 gene, according to 30 months prospective cohort study results. (A) Three survival curves were drawn for each genotype. (B) Two survival curves drawn assuming a dominant model of 'A' allyl. The result of analyzing the difference in the formation of a DNA-nucleoprotein complex due to the difference in the bases of SNP rs2282691 'A' allyl and 'T' allyl by EMSA (electrophoretic mobility shift assay). Lane (1): The C / EBPβ olivonucleotide probe corresponding to the 'T' allyl of SNP rs2282691 was applied without adding the nuclear extract. Free probes were found outside the figure. Lane (2): The C / EBPβ olivonucleotide probe corresponding to the 'T' allyl of SNP rs2282691 was mixed with the nuclear extract and applied. The position of the DNA-nucleoprotein complex is indicated by an asterisk (*). Lane (3): A C / EBPβ olivonucleotide probe corresponding to the 'T' allele of SNP rs2282691 was applied by incubation with a nuclear extract and further anti-C / EBPβ. The super shifted band is indicated by an arrow. Lane (4): In order to confirm the specificity of the band by a competition experiment, an excessive amount of an unlabeled SNP rs2282691 corresponding to the ‘T’ allyl of C / EBPβ and a nuclear extract were applied. Lane (5): The C / EBPβ olivonucleotide probe corresponding to the 'A' allele of SNP rs2282691 was mixed with the nuclear extract and applied. [Sequence Listing] SEQUENCE LISTING <110> YAMAGATA UNIVERSITY HUBIT GENOMIX, INC. <120> Polymorphisms in AECOPD genes in patients with Chronic obstructive pulmonary disease, and use themselves <130> 0601 <160> 5 <170> PatentIn version 3.1 <210 > 1 <211> 20001 <212> DNA <213> Homo sapiens <400> 1gagataatct gtttgtttgg gacaaagtaa gaaaaaagaa caagagtttc tgcctggtag 60tccagagaat tcttctggat tttatccaag accaccaaag tggtacacag aaaaccacag 120aataacattg taattgtggt gtgtaaacta tgcataagtt acgtagaaag acaaaaagat 180gaactgatcc aaatatctgc aacaactttt gaagacatag atagtacaat aaaatataaa 240cagaaacaac agaaagttaa aaagtgggga gatgaagtta aaatgtagaa gttttatcag 300ttttctcttt gtttgtttat gtaatcagca ttaaattgta atcaattttt ttaaaatggg 360ttataagaca ctatctgcaa ccctcatggt aacctcaaat ctaaaaacac acaacagata 420cacaaaaaat aaaaagcaag aaattaaatc ataccactag agaaaataga cttcaccaaa 480aggaagatag aggagagagagagagagagagagagagagagagagagaga ggag taagtcctta cttattaata ataacattaa atgtaaacag 600actaaagtct ccaatcaaaa gacgtaaaac gtggctaaaa 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1500aacaacctga aaataaacgc ttgaagcaat ggatatccca tttactttga tgtagttatt 1560acacattaca tgactgcatc aaaatatctc atgtaaccca taaaaataca cacctattat 1620gtactcacaa aaattaaaaa ttaaaaaata agaagcctac agttaatgaa gttgaaatgc 1680cagaaattct ctggaatgac aggaaggagt ccagagactc agggaagtgg atctgttgga 1740atggatctag tctatctgtt ggatctgttg gaatggatct agtctatgtg caagctgctg 1800ggaggcccag ggggacactc ccttcaatcc aggtattagg aaatgttttg atgagttgtg 1860ggaggagggc aggatgctta agagctctgt agtggctatt ctttatagat cagtgatgac 1920agtgggacat gatataatcc catagttctc aaacttttgt gtatttcagg atcacctaat 1980gggcttgtta aagcagtctg ccatgctact actggtggta attaaagtaa tcatgatatg 2040ttgacagatc ctattcctgt gcccctttcc atggtgcacc taaaacatac gagcaatcca 2100atcccagaat cccatttgga agttctattt tatggggcca attcaatgtc agtaagtgtc 2160ccagcaagaa atagataaca cttaaaatgg aagacttgga ga aaggttta gtaaagaagc 2220tatttacaaa gatgtaagtg ggtgaaaaga gaccacaggg gtgtttggta ccatgaggct 2280attaagaaca tggttagact gcaaaaaatt tctccagttc tgtaggatat ctgttcactc 2340tgatgatagt ttcttttgct gtacagaagc tctttagttt aattatatcc catttgccaa 2400tttttgcttt tgttgcaatt gcttttgaca ttttcatcat gaaatctttt cctgtgccta 2460tgttttgaat ggtattgcct agagtttctt ctagggtttt tatagttttg ggttttacat 2520ttaagtcttt aatccatctt gatttcattt ttgtatgagg tgtaaggaag gggtccagtt 2580tcagtgttct gcatacggct agccagtttt cccagcacca cttattaaat agggaatcct 2640ttctccattg cttatttttg tcaggtttgt caaagatcag atggttgtag atgtgtagtc 2700ttatttctga gatctctatt ctgttccatt ggtctgtgtg tctgtttttg taccagtacc 2760atactgtttt ggttactgta gccttgtagt atagtttgaa gtcaggtagc atgatacctc 2820cagctgtgtt ctttttgttt aagattgtct tggctattcc actgcggcct ctcagggagt 2880gagggaaagg gagagcgtca ggataaatag ctaatgtgga tggagcttaa tacttgggtg 2940aggggttgat aggtgcagca aaccaccatg gcacatgttt acctatgtaa cagacctgca 3000catcctgcgc atgtatccca aa atttaaaa taaaataaaa attttttaaa aagaacatgg 3060ttatattcct aaatctaaaa aggaaaaaca ggggatagct cagcaaacct ggataggaga 3120gtcatggaga aaccaccttg aaagaagtta aaaccattag ttgagggatg tatccaggtt 3180gaggaacaca gggagacaac agaggagaag aacctcaact taactcttct ccctgctcta 3240atgtcctcag aagctccctt gtgcctaaac ccagtcagaa gctagaaggt cagcaacctc 3300actgatgtaa cccaccaaga ttggcttcct agggcagaga atagaatgga aaagaatgga 3360atgtgtgtct gaagggacaa gcttataata tccttttctc aggtgagtct tgacacattt 3420ttgtgtcaaa gttaggtagg tctcaaaatg aattgagtgg tgtacaatct tttgatattc 3480tctggaagag attgcacaag attgaaatga ccacttcctt cagtgcttgg aggaattagc 3540tgtttgtgcc ctttgagccc agagaatgtt ttgtgggaag attttcaatt cctgattcca 3600tttctttaat agctttagaa tgactttcta ttttttattt atttgtgcac cctggtcagt 3660tagattttta agaaattcat ccatttcctc taaactaaat ttttggcata aggttcttat 3720cattttcttt ttggtgtctg caggctctgc aactggttat atgtaccttt ttcctttttt 3780cttttctttt tttttttttc tgggacagag tcctactctg tcactcagac tggggtgcag 3840tg gtgcaatc ttggctcact gtaacctcgg cctcccaggt tcaagcaatt ctcctgcctc 3900agcctcccaa gtagctggga ttacaggcat gtgccaccat gcctgggtaa tttttgtatt 3960ttttttagta aagacagggt tttaccatgt tgtccaggct gctctagaac tcttgacctc 4020aagtgatccc cccgcctcag cccctttttt cttttttttt tcattttcag ttcaggagag 4080ttttagctta attataggct acagaaccag ctttgggctt catctatcct ttctaatatt 4140tactgtttcc tatttctcta atcctagctc tttatttctt ccctttactt tcactgggct 4200tattttgcta tagtggaatg cagagggatg agtattccag gaaggcacaa aaactgtgcc 4260aagtcttgga gctagggatg agtgggaaag ggacatgttc aaccatttta agccattccc 4320tccccacctc ccagctccca gatatgtgcc cctcgcagga ggagcccagg aatgggccaa 4380acacctcact tctttgctct gagggccacc ccagccctcc catcaacagc tctagaaacc 4440caatggtcct tcctggaaac acggggcctg catcaatcag aggtgtttga accatgtccc 4500tctgggcctg aggggcagaa ggggacacaa tatgtaatgt aaggagcccc tgtcatcaga 4560aatctgactt aatctgtttc agatattaga cttccacata aaagttgact tggaaaaaga 4620cttctgctgc taaacaaaag ttgaaactgc cttggtgata aaatataag c agaccagctt 4680tctcttctag ctttccctct catttcccat aagattttgg tcaagttatt taatctctct 4740gcatccgttt cctcttctat gaaatgggca tgataataat ggtatgtacc tcctcaaact 4800aaatatataa cgtgaacaga gtccttagca cagcactctt tctctacagg agttaatttt 4860cattgttttt ctctttcctg ttggagaaag taagaagaaa acagctcctt tatggcttcc 4920catggtgaat ggctggggcg cgtctgtgtc cctttctcct ctctggctcc ttgtggcctg 4980aacagccaga aggaagccat gccatgctgt ttcagccctc agcttccctc ttgcatttcc 5040tagaaaagtc tttggtgccc agctccagct cagcagattc aggatccccc ttcatcatga 5100cttggtcaac gccctgctca ggccaaggtc ctctgagagt tccaagcttc tccactccct 5160ataaaaggcc ggcggaacag ccagaggagc agagaggcaa agaaacattg tgaaatctcc 5220aactcttaac cttcaacatg aaagtctctg cagtgcttct gtgcctgctg ctcatgacag 5280cagctttcaa cccccaggga cttgctcagc caggtaagtc acctcccttc gactctccct 5340ctctttccct ctgtttctct attcaaggaa gacctaagcc cgagtgctcc tccacttttt 5400ttttagattg agtctcatta tgttgcccag gctgaagtgc aggggtgcga tcttggctca 5460ttgcaacctt cacctcccag gttcaagcg a ttctcttgcc tcagccttct gagtagctgt 5520gattacaggc acccgccatc acgtgcagct aatttttgta tttttagtag agaaggggtt 5580tcactatgtt ggccaggctg gtctcaaact cttgacctca agtgatcctc ccgtctcggc 5640ctcccaaagt gctgggatta caggcgtgag ccaccaggcc cagccaagtg ccccacttct 5700aagcccacca gaatagtaag gctcctcaga ggttcacttt aacatctaat tttaaagata 5760gaaagctgaa gcccatgttg gaggcagaag ggaccctagc catccacctc caggttattg 5820cagagcaaga atgaaaccta agcttctgac tccagattta gggccttttc tttgacctca 5880tctgatcgtc ccaaactctg cagatctggg accacaccca ggacctttcc cactggcctt 5940gcccgtggcc tcccctagat ggctgtgaca tgtctccacc atgcagctga gcctttgaga 6000tcctgaggca catgtcacag gtcccacctc acctcagggt ctagggtggg agtgctgggc 6060ttgggggtga gtaagatcta tttcttcctc tttgctttgc atcccataca gatgctccct 6120gctgtattca agctgagaaa agcctaacac atcctcaaag tctttttctt tgtaactatt 6180tctagatgca ctcaacgtcc catctacttg ctgcttcaca tttagcagta agaagatctc 6240cttgcagagg ctgaagagct atgtgatcac caccagcagg tgtccccaga aggctgtcat 6300gtgggtaga a aaatccctgc tcacctggct cctccccact cccacattcc ccaatccaaa 6360gttctgcccc aggagacaga cgtcagactg acttgagatc ttaggatgag atctagccag 6420actgtgtgat gcaaatcctc caattttggc tgcacaacag gtccaaagag gacctataat 6480] ttcccacacc ttgtttcctg gatgggcacc agcccacacc ctttagcaga tgccaggatc 6540agtttcccag gggcagcaag agcagtggct gcctccagag accccttctg tccacacacc 6600tcctacttcc tgtcctggag gggtgcccct tcacctgtag taggtggacc aggcaggttt 6660agaacccagt gtgtcatctc ctgggtaaac cctcaaaggg ttccatctaa ctgtgccaga 6720tctccttcct ccacagcttc agaaccaaac tgggcaagga gatctgtgct gacccaaagg 6780agaagtgggt ccagaattat atgaaacacc tgggccggaa agctcacacc ctgaagactt 6840gaactctgct acccctactg aaatcaagct ggagtacgtg aaatgacttt tccattctcc 6900tctggcctcc tcttctatgc tttggaatac ttctaccata attttcaaat aggatgcatt 6960cggttttgtg attcaaaatg tactatgtgt taagtaatat tggctattat ttgacttgtt 7020gctggtttgg agtttatttg agtattgctg atcttttcta aagcaaggcc ttgagcaagt 7080aggttgctgt ctctaagccc ccttcccttc cactatgagc tgctggcagt gggt ttgtat 7140tcggttccca ggggttgaga gcatgcctgt gggagtcatg gacatgaagg gatgctgcaa 7200tgtaggaagg agagctcttt gtgaatgtga ggtgttgcta aatatgttat tgtggaaaga 7260tgaatgcaat agtaggactg ctgacatttt gcagaaaata cattttattt aaaatctcct 7320acacagtggt gttttcttca ggagtaactg ccaaccagta ggggctctca gaggtgtggg 7380tggatggcat gccagtggaa ggcagatgta cagaggccct ggccatgggg ctagcctggc 7440tcatctgagt ttcaaggagg gcctgcctca ccagcccagg ctgttctgga acaatggctc 7500cagtccccag catgggcgct cctggctcgc aactcatccg tttcatattc agttcatttc 7560tttctttaaa accacactcc tgccaaaagt aaacccatcc ctccccctct agtctcccac 7620cccaatctat gcctccccac tccaagcagc aagtgattca gacttcatgt gcacctgaca 7680ctcagaggga aaccagatga ccaagtccta gtgagcctca ggattggtag ggaagagggt 7740gcttgggcct aatggacttt agaggatggg gaagtatggt catagcaaag attcttttag 7800gggaggtcca agatagcaat gtggggagag ggcctggggg gaatatcccc aaacggtccg 7860atatgggctt ccagaatgta aaagttggag acatgtagaa atgaaagttg agggcaggtt 7920ctagaggaat tagaatatca gggtggggag ttta gactca gttctgaagg caggaggaag 7980gtgcaaaggt ttttgagtca tccagaggga agaatggatg ccaggaagac tagctattgg 8040ctgtggataa gatggagttg agaagggagg gcggaaaaca gcctactgca atagtccaag 8100caagagaaaa taggggctga agaagggcag tcatgggggt ggagaagagg aggcagatta 8160agaagactgg gccagatgtg tgacatgttg aatggggaat gagggaaaca cagcatgaga 8220cctttgccct ctaaaagtga tctttaaagg cgtgccaacc ccaagaccta gaaatacttc 8280cttccttggc tattcttcct tgaatcacac gttgtacaaa ttgaagcttg tgattgtccc 8340ttttaaagcg ttggccctcc tcttcctata tgtccaccaa gggtttatcc tgctcttccc 8400aagacagcct cactcctcca gataagctgg gtgggcatct caatcagacc cagaacctac 8460ccatgcctgg acacccactg agatcttcct caagaagaga tgggcactgc tgacaacagc 8520agggcctgtc ccccagatct ggtaccacac tgcatcaggc agtacacttg gcaatgtggg 8580acacctggag gagttgtcct aggaggcaac agctctaggc tctaatgcca gatctgcccc 8640tgaattgctg catctctctt tagccctcag tcacctcatc tataaaatgg gtttagcaac 8700attttacagg cagacaacct cacatgatcc cctccagcta gggcagttgt gatttggtga 8760taacatcacc cccc aagccc tctgggcagg gcagggagaa gaagagtcct gcacccaaat 8820gtggatccag ctgtgaaccc cagctttctg ttccactgta ccactcctgg cagccaaatc 8880tggaggcaga gaatctgcac caccaccctg actcaaagct tacaccccag gacaaaggca 8940gtcactgaga gctagtgagt cacttattag cccagttccc ctggcaccag gtgcaacatg 9000tctccctcct cctgacccat ccttcagccc ctggctccca cctctcaatg ccaatcaagt 9060tttcactctg aaatccaaga gacccagagg gttgggggtt gatgattgta taatttaaat 9120gtttaaagtg caacaacata gcttatcaag accaagcaga tcctctgtga cctagcaaaa 9180gcagggcaga aggaatggtg tagggctggt agtttcgggg acaggtgaag ccatgtggtt 9240tccagagccc acaatggaaa gaaatctgct cattttcttt ttgacgggca gtgcctcagc 9300atttttctgt gcctctgaac ccatccaact gtgtccaagg cgcaggcctc tttgcctctc 9360ttcagcttga atctgtgaac agagaataag agagaaggct gagccaccca agccaccact 9420gggtgggcaa cgcacaagcc ccgcctcctc cggctgccag gtcccctaaa ctgctcttgc 9480taggccttgg gctcccctct gtaaatgaag ttgctgcaca ggatgtgtgg tttgcacgct 9540ggggaacagc caaagcacca ggagcttatg aattgaattt cctggacccc tctcctggtt 9600tcaatttggt gtatcagggt ggggctgaac agcctgaaaa cattctgacg attaggtgag 9660atgagaaatt cccatcctgg aatagctctt tgcaccaact ttaatgtttc tgtgtccatc 9720tggggcctta cttgttgcgt tggactcatg tgtttgtgtg tgtgtgtgtg cgcgcgcctt 9780gtgtgtgtgt gggtgtgtgt atttttctct tctgatctgt ctacttccta aactggacta 9840ttgtccaatc tatagaccaa ctctatctct tactctctgg ttgatcttag acaaatatct 9900ctaccttcct gaacctcact tttcatgata acaaaaatat gtgccccaag aggatcgaca 9960gagagaatat acatgaaaaa gccttaaaat actgactggt ttgtgaaagc tactccaatt 10020aagtttgtaa agaccctgtg atacagctat gtttatgacc cttcacagat aaggaataca 10080gaaggataca cagacaattc agtacaaagg agttacctgt ccacggtcac ccaagactat 10140gggttccaaa tccaggggca ggaactcatc ctggttccct tgctctttcc agtgcacaat 10200aacccctcat gcttttgtta aacctctgag ctcataatgc tgggatacaa tacttggcac 10260cttggagttt caggaaacac aagccactgc aggagcacag atgcatcctt cctcacacct 10320gctaggaagc tcagcgctcc ctctagcgcc caagtgtggc ttgcacccag gtgctggcag 10380ctctgggttt agaaacctta ttctggtaaa atactgtcat ctagtgtcta ctcttgtaat 10440tccaggctgg cggggttctg ttctctaggg agagattgag caggtgatca cttacattaa 10500gccctcattg gagcagatgg agctggtatt tctgtaacac aggattgccc tcaggggaat 10560ctcttgctcc gcaaatgaga agcaacatct ggagaagggt acctgcacta gaagaggaac 10620acagacgatg gtttgcatcc atttctcaac ctctcacctg tagcacaatt tttaaaaaac 10680aaacaaaaaa acgcctccca ggacaagccc tggcttgggg accacatcca gacggtgccg 10740gcatagctct gcctcatcag ccaccttgtt ctggggctag gtaaagagtc agaagattgg 10800ggttcagatc tgagctcccc gcaccccagc tgagtggccc tgaggaagat gtgccttcac 10860cctggcccca gacttcccat ctgtaaaata aaactttttg gagcctggtt tctaaaggct 10920catccagttt tggtatttca ccacatgtgc atggttctgc cccagtattc atgtccaagg 10980gctctggacc ccacaccctg gggacctggt tagtctgatg caggacacag tggtccacag 11040ttcacagacc tgatctgggg aagattagga attgttcgag gagtccaaac tgatgagatg 11100acaagggaag agctcccagg aactagctag ctgccctact cagtctcttg aatcccctcc 11160tgccctggtc cttagctctt tcccagcccc caacccccac tctgtccatt tgtgcattct 11220cctcccacac cacatcccgc aagagacctg cattccaatc ccagcttctt cctcctagtt 11280tttctgtttg cacccccttt catgcctctt actaggttgg tattctccaa acagtcttgg 11340agggcagaca tcatcaactc catttcactg atagagaaag tagagtttga gagctaaagt 11400gacttgccca agagtgacca attgttagag cccggtcttg ccttctgact ctacttggtg 11460tctgtcacac tgtagaagct acacccaggc tgtcccctgg accttaggcc tcccagcttt 11520acccgagtaa cctcacaggt tcccttcctt gggtctggat gaagcagagc ctccttgtac 11580tgaagctgtc agttcagttc tctgtgtttg ccccaactca tggttctcct actagccctc 11640accccagcct gtccaagttc accattcctc ctctttaggt aggaagaggg gagatagagt 11700tggtgagttc aaggagaagc ctgctccaac cccagagtgg ctgaccacgt ttctgccctc 11760cccagagaga agcaaaatga tgcctgccac actcactgct cttgctgtcc acatcttccg 11820gccacatccc agctagcagc aagcacacca gggctgtggt gatgatctgc atgtcttctg 11880gtctggcttg ggccttcctg gagcagcttt gatgagcctg gtgaagctaa gagctcacca 11940ctgtgccgtc ctgcaatgct gagggctttt atttgaaaaa ctccacccaa aactccaggc 12000catggtggga gccacaagtg ggaagccata gggacaggga acagttgctc acacccagcg 12060 accatgtggt aaactccttt ccccacctct cctggctggt attcgtggtg gtgatggatt 12120gataggttgc attgggaact tccctaagtg tgggagcaga taagaatctt gaaaaagtgc 12180gtgggaactg tcctggttga gtgggaagca aagaaaagta agtgagagga agtggcctct 12240gtgtggcttg tccaacccta cgtcattcca tccaagtact actgcttcag ggcagaagag 12300tctggacctg aacagagata cacctgcatt tgaatcccag ttctgcagag gagggattac 12360ataaacttac ccaaattcat attcacacaa aatgcaaata gcaatacatg tctcaaggac 12420gtgcgataag aattaaataa cattctctat gtccagtgac tggcctagat atttgccatt 12480tctatttttt ctattttttt aatcctcaac aattttcaat agcttattct gttctaggtc 12540cttggttagc cccagagaag gcagttacca atgaggagca ctgcctgacc tcaagcaact 12600tctcttcaat tcacagataa acaagtgaaa gccacccagt gtaagcacag agatagatgt 12660gttatgtttg ggttacagag aggactgaga gctactcagc agcatctgag agtcctgcca 12720ggggaatttt ccctttttgt tttttttttt ctagagtctc actctgtcac ccaggctgga 12780gtgcagtggc acacactcac tgcagcctca aactcctggg ctcaagcaat cctccagcct 12840cagccaggga atttttcctt tttgtttttt tttttctagg gtctcat tct gttgcccagg 12900ctggagtgcc atggtgcaaa ctcactgcag cctcaaactc ctgggctcaa gcgatcctcc 12960cgcctcagcc tcacagtggg tggaactata ggtgtgcact accttgccca gataattttt 13020ttatttttta tttgcgtaga gatggagtct cactgtgttc cccaggctgg tcttgaactc 13080ctaggttcaa gcaatccttc tgcctcggcc tcccaaagtg ctgggattac cagtgtgagc 13140caccacccca gccaggagaa tgttctttaa gcaatatctc ctgtgtgctg atgtggagag 13200gggaaagcta agtagaggca gggtcttcta tgaggacaac aaggcataag ggagcacact 13260tcatttcaaa tcatcaggaa aagcagaagt gtagggtcca aggagagtga caggagtgag 13320agagtggttg cagagagatg tggctgtaca gctacagaga agactgggtc ataaagatgt 13380atgcagtact cggaataaag agcacatagt aggtgctcaa gaaaaagtag ctgagccaaa 13440tctaggcatt gaaatcatga agcctgtgtg cattatatag agtgatggct agactgatct 13500actctgattg tgcagtcttt agctcaaaaa cctgcagtgg ctccacacca tcaattgggt 13560ggagcaccga gctactcacc tggatatttg tggttcttca tgtcatgcct cagtggaggc 13620cagcacattc gggtaattgc aattcctgat tgcaagtaac tgccacttgc tgtactatct 13680ctgggatttt gctcctgctg taacctc cac ctgcagagcc cacctctctg tctcactctg 13740tgaacatcct acccattgca aatgtcattg tctccacaaa ggcacttctg ccatatctag 13800cagataggat ctcagcctct tgtgagaact ctcatgcctg gattgccata caattcctgc 13860aacactttcc tttagtccac tctccaaaca tggaaatgtc caagttgaaa gggctcttcc 13920agactatcta caacagtggc tcttgaagtg ttttgggaga gcttttgtaa aaatctgaat 13980atcttagtcc catttcagga tgagaatcaa aacacatgtg catgtacatg ttggaaagaa 14040aaaattttca ggtaattttc tcagacacca gaagataaga atcacatatc tcgtataatc 14100acttcatatt atgatgaaat gagaggaaag taacttgtcc aaggtcatgt tgacctcagg 14160gacaatggaa cttgagccct ggtcttttga ccttcatgtg cagctgcctc tgagaacaca 14220ctttttgcac agtccacaac taacttcctc tgctcagccc aaccaagtac catttatcct 14280ttcaactgct tcctgccacc tgaaaagtgg ttgaaaccac aaactgctgt tgtttctatc 14340ctattctctt tgtccttaga gatggataaa tactatgtgc tattcatcct actatgcgct 14400atgcatccat ctttttcttt caacttttat tttaagttcc agggtacatg tgcagggtgt 14460accattttct ttatccagtc tatcattgat gggcatttgg gttaattcca cgtctttgct 14520attgtga aga gtgctgcagt gaaggttcat gtgcctgtat ctttataata gaatgtttta 14580tattcctttg ggtatatacc cagtagtggg attgctgggt caaatggcat ttctacaaga 14640gatgaatact tttttattcc tttaccatag cgtgtcagga gagagagaag atgcaagtgg 14700gtggtcagtc agccaaatgt aactgtaggt cctttagatg taggaaacag acagggggaa 14760atgtggccac cttggaaatt ctttgttctt cccctcctcc tggggttgct gcccagccca 14820atcaagaagg gaaaccgggc tgcccactcc accaccccct tcccctcccc ccacctccct 14880gtggccacag tcagaccagc acaggcaaaa agagaacagc aagatttagt catgcttttc 14940ctttccaggc tccctcagca catggaagga actgactgga ggctcttatc acacaggaac 15000ttaatgtttc tcatgggtat ctcttccacc ccaatgtgac tgtcaaggac acagacttct 15060ctaggctcta gcacaaaggc taggaccaag tagtaactca attaaacagt tgtgtaactg 15120aacaagctgg aacagatggc ataggatgga ctgatgtata agagatgctc tcagaataaa 15180cactgaacca gttattcata tgaacacatt tcttcgttag aaatttgaaa atcagaactg 15240cttggagata ggttttctta gtcatctttt atgaaataag tactgattac agaggacagg 15300tagagaccag atagagttca gcccatgcag ctgcctctta agacatctcc tcc agaggcg 15360cccagtggtg tctgagggtg tccaggagtg ttccagccct ctttcctcct cctggatggc 15420ccgagtggcc tggggcttga ccctgcctgc ttgccttgca gttagagttc agcctgatcc 15480ttgcagatca gggcatagaa actgacaagg gagaagtcat ttgcccagca acatacaacc 15540acagaaatac tggtcttgct ttgtccagct ctggggtcaa gccttctgag ttccctcact 15600tagggaggaa gcaagctata gaggaaatgc aggatagtca gacacaactg ggtcagatct 15660cagctccacc gctagagcct aggcaagtaa tttacacttt ctgagtcttt gctctctcat 15720tgtaagctcc acagctagct ggacttggca caactctcct cattgttttt aagataaact 15780ccccaggctg aggcaggaga atcccttgaa cccgggagac agaggttgca gtgagccaag 15840atcgcaccac tgcactccag cctgggtgac agaacaagac tccagtctca aaaaaataaa 15900gtaaaataaa ataaaaatca ataaatcaat aaataaactc cccaaacctt aaacacattt 15960atttgaggtt atccaaagga tcaagtgaga tttaagagga gggaggggag agaaaaagaa 16020aaagttgaat cattcaaaat cattgtggag caccgaatat gtaccagaca ctggggatac 16080cgcagcggga caagacaaac atgggccctg gtattgggaa ttgtggtaag tcaagattag 16140tgcttcgagg agacggacaa tgtcctgtga gct gcagagt gtgcggaaat gctcaccacg 16200gggccggaca catggtagat gctcaatgaa actgtgtcct tttcccctcg atcctggacc 16260attctcagca tcagcacatg aacccaggca gtaccaccct ctcccctact ggggcccatt 16320tcctggtctc tttcccttct catctggtgc tgcttgaaac ccacaccaag ccttgtttca 16380ttgcccacat gagcagggtg tcctcttggg tcctcaagtc tgcaccagac cccctaccta 16440ctgcaagcac aaattcaaat ccacagcaag gacccgagca ccaaaaaaca cctcgggatt 16500caaagccctt tttcatgttt tccagcacct ccttatactc tgcccacatt ccctttcccc 16560ctatgttttc cgctgacatt tagcactttt tttttaagga ggcctctcgt atttacatat 16620cagaaacaat gtgaaggagg catggagaac ccctcatggc aacaggaaaa ctgaggggtg 16680ccccaaagga tgtcacaggc acccagcttt tcaatgtgct gctgattctg acacaacatg 16740gcccaccaag ctcagggtga catgcaactg acttggttgg tctcacaggg tcctgcacag 16800cacagtcggc agacttgcag ccacacaatc ccgcttgttg tctgcacata cactgggatt 16860tactgcccct tctgcctgga acactctttc ctgctttatg tgcctaactt cttgcaggct 16920gtccaactca gagctcccaa agtgaaccca gcccactcct cacagcgggt atctcacact 16980agtgtaattt cgt ggtggtt cacactcctt cctgtctaga agctcccaga gagcagaagt 17040ccctctgcta tggtcagcat ttcatgctgc agcgtttaac actgcactga tgtgcagcaa 17100ctattagctg gaaaagagag aaggagagat tttaacatca ctatggctga gctcatgtca 17160cggaggagaa atgaagaccc tggaggttag cagacagaca ggagttcaca cagattgcct 17220ggatcagaat caggataacg agctctggcc aaaatcctgt gcttggttat actgcactgt 17280gcagtagaag actgcaattt ccatgtgtgc taaatgggca ccatttgaca cgtaagattc 17340cagtgacaga ttgcagttca acagtgtctt cattcattca ttcattcatt cattcattca 17400ttcagcaaac acctttcagc tgggcctgca aggatgaagg catgatttat caggagagga 17460gaaggaacag gggctagcat ggtgagcagt ggttatgagg gtgcggtctg aagccagaat 17520gccctggtgc ccagtcccag cttccctgaa cgctatttgc gtaacttctt tgagttactt 17580aaccactgag cctcatctat aaacagggat aataatagca tctgcttata ggcttgttgt 17640gaaaattaaa tataaagtac ttagaatagt gcctgacata tttctccggg aaaatgggaa 17700cagtgacatc actgtatgaa agtgtgtagg ctgctgtctt cagagcttgg gggtgtgtga 17760ggctgcatgg caaggggtgg ctagaaatga agggtgagta gggctgggtt cagttgtgca 17820agatcctgtt tactgagctt tgccttgacc ctgttggcga tagagggcct gagctaacct 17880tttgctcagg acagaggatg gcccgatatg tggtgagggc tgagggctgg tgccaaaggc 17940taggacagat tgaaaggagg cagccaggtt ggggaggctg ggaaagagct ccagtgagag 18000atgaggaggg ttgaggttgt gtatgggagc atgtgaaaat caggatatga gctggctctg 18060ccacctgctg tctgtaggat ttgggcaagt tcctctctct aagcctcaat ttccttaccc 18120aaaaagtggg aaggttggcc taaatcagcg actcttgccc ttgaaatagg attcttatat 18180ggatcattca ggaagctttg gtcaaaagga cacatgcttg aatcccgctc tagatcccca 18240tattctaaat gcaggtcagc cttttccaaa tgaaaaggct tctcatataa tccaatccca 18300ccatttctct accctgtcaa aagcccctaa cctggatggc cactgggaca ctctctagct 18360tggcatttat tgactctcag gatctccaag caagggcaga ctcctgccca ccagccatgc 18420tcaaagcacc acacttgccc attcctgctg ggaccggcag tcggatctgg cctgttaaca 18480gcaaacactc atgcggaggg ggcctgctgg ggaggttacc agaaatgccc cttccccttc 18540tgatgaggct caaaggcagt gaaacctaat gggactgatc ctggcctttg aggtatttag 18600gtccctcacc cactctgaga accacacttg caatccttcc atctgaaact tagcaagcac 18660cttgaactgg gaatgtgttg tgaactctgc actgtggttc ttgagctgtt tgatgctgta 18720tcaaaaatat tcaccttgca acatctctgt gagtatgcaa ggggtccctg gggtcaggca 18780tacgccatat acagcttggt gcccaggagt acaccggatg aaagtgtcct ttcatcttct 18840ccctcctggc ccccactccc gtagagcagg aagagccctt aggaagacct ggggtagacc 18900gggtcatatt acagatgagt agcctgaggc tgagaaagaa aaagtgaact gtcatgtggg 18960gacttggtaa caggaccctg gagacagcct tcctcttaat tcccaggcca ggctctttgt 19020cttgtgattt gctggccatg tgctgatatc tggagggctt gctatggaca gagagaaggt 19080gaggatgctg aagttctgca ggattcccac agcccacctg gaggccagga cacagtgact 19140ttgcagggag tggtgggggg aaagccccta cattttcacc ccacagagca aagatgccag 19200gggcaggaag tggcaccact gacttcaaaa tatgcccact aaaaccaaca aggccaccca 19260tcattgaggt gacacaccca caggctactg ggaaagtccc ccaaggagtc cttccctttc 19320tggggtggca ggtggaacag cctgggacac tccatggctc acagcccatt tcccaatgca 19380tcgtaggccc acagtcctgg gattccatct gtccatgtct ctcaggactg gcccacttcg 19440taagaagccc ccgtcacctg gtctggcctc ctgggctctg gagtgaacta aactgaccac 19500agatcccgca gtaagctcaa actgctcaac ctcagcaact gaagctgagc ctcagacaca 19560ccatcctgca cctggcctcc tggccttctt ctccatcctc tgatggcgtg ctatccagac 19620atccatattt taatccaggt tgccacccat tagtacctgg tcaattctgc ttatcaccct 19680aaacctctga gtcatcatct gttaaactgg gtttacaatg cctctgtctc aggaatgtcg 19740taaggactaa atatggaact acatgcaaag gctttctagc acagagcctg gctcagaggt 19800tgtgatttct ctgtgttctg tgtttcctcc ctgcctgcgt ggtttgcctg aggcatcttt 19860ataaatgctc tcaagagctg attctaagtg agtcaggtcc tatttctcag ctgtggctgg 19920ctagtattgc ttccttagaa gagtgtgagt ttcaggaacc aagtttacaa gggagcacct 19980gaagtcagga ctgctgcctg c 20001 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <400> 2attggagtag ctttcacaaa ccagtcagta t 31 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <400 > 3atactgactg gtttgtgaaa gctactccaa t 31 <210> 4 <211> 31 <212> DNA <213 > Artificial Sequence <400> 4attggagtag ctttcacata ccagtcagta t 31 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <400> 5atactgactg gtatgtgaaa gctactccaa t 31

Claims (21)

  A method for determining the presence or absence of susceptibility to acute exacerbation in chronic obstructive pulmonary disease, which comprises detecting a mutation in a CCL1 gene or a DNA region near the gene in a subject.   The method according to claim 1, wherein the mutation is determined to be acutely exacerbated in chronic obstructive pulmonary disease when it results in a decrease in the expression level of the gene.   The method according to claim 1 or 2, wherein the mutation is a single nucleotide polymorphism mutation. A method for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease for a subject, comprising the following steps (a) and (b).
(A) A step of determining a base type of a polymorphic site in a CCL1 gene or a DNA region adjacent to the gene in a subject.
(B) A step of determining that the subject is susceptible to acute exacerbation in chronic obstructive pulmonary disease when a mutation is detected in the base type of the polymorphic site determined in (a).   The method according to claim 4, wherein the mutation is determined to be acutely exacerbated in chronic obstructive pulmonary disease when it results in a decrease in the expression level of the gene.   The method according to claim 4 or 5, wherein the polymorphic site is a site on the CCL1 gene region and is a polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1.   The base type mutation is the one in which the base type at position 10001 in the base sequence described in SEQ ID NO: 1 is mutated from T to A at a site on the CCL1 gene region. The method described.   The method according to any one of claims 1 to 7, wherein a biological sample derived from a subject is subjected to a test as a test sample.   The method according to claim 1, wherein the acute exacerbation susceptibility in chronic obstructive pulmonary disease is the frequency of acute exacerbation in chronic obstructive pulmonary disease.   The method according to claim 1, wherein the acute exacerbation susceptibility in chronic obstructive pulmonary disease is the severity of acute exacerbation in chronic obstructive pulmonary disease.   The method according to any one of claims 1 to 10, wherein the acute exacerbation in chronic obstructive pulmonary disease is an acute respiratory tract infection in chronic obstructive pulmonary disease.   A drug for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to DNA comprising the polymorphic site according to claim 7.   A drug for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising a solid phase to which a nucleotide probe that hybridizes with DNA containing the polymorphic site according to claim 7 is immobilized.   A drug for determining the presence or absence of acute exacerbation in chronic obstructive pulmonary disease, comprising a primer oligonucleotide for amplifying the DNA containing the polymorphic site according to claim 7.   The drug according to any one of claims 12 to 14, wherein the acute exacerbation in chronic obstructive pulmonary disease is an acute respiratory tract infection in chronic obstructive pulmonary disease.   A screening method for a therapeutic or prophylactic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising selecting a substance that activates the expression level of the CCL1 gene or the function of a protein encoded by the gene. A screening method for a therapeutic or prophylactic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising the following steps (a) to (c):
(A) a step of bringing a test substance into contact with a cell expressing the CCL1 gene; (b) a step of measuring the expression level of the CCL1 gene; and (c) the expression compared to a case where the test substance is measured in the absence of the test substance. The process of selecting substances that increase the level A screening method for a therapeutic or prophylactic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising the following steps (a) to (c):
(A) a step of bringing a test substance into contact with a cell or a cell extract containing DNA having a structure in which a transcriptional regulatory region of the CCL1 gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene Step (c) A step of selecting a substance that increases the expression level as compared with the case where it is measured in the absence of the test substance. A screening method for a therapeutic or prophylactic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising the following steps (a) to (c):
(A) contacting a polynucleotide containing a C / EBPβ recognition sequence and C / EBPβ protein with a test substance (b) measuring a binding activity between the polynucleotide and a transcription factor (c) absence of the test substance A step of selecting a substance that enhances the binding activity as compared to the case measured below   20. The screening method according to claim 16, wherein the acute exacerbation in chronic obstructive pulmonary disease is an acute respiratory tract infection.   A preventive or therapeutic agent for acute exacerbation in chronic obstructive pulmonary disease, comprising as an active ingredient a substance that activates the expression level of the CCL1 gene or the function of the protein encoded by the gene.