JP2006333811A - Polymorphism relating to weight loss in chronic obstructive pulmonary disease and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene relating to weight loss which is the most important prognosis determining factor in COPD (chronic obstructive pulmonary disease) and a polymorphism relating to weight loss in COPD and existing in the gene or a neighboring DNA region of the gene. <P>SOLUTION: In order to solve the above problem, the inflammation inducing characteristics of sPLA<SB>2</SB>(secretory phospholipase A<SB>2</SB>) were intensively studied in relation to the chromic and systemic inflammation which is a latent factor to cause the weight loss in COPD patient, and 12 single-nucleotide polymorphisms (SNP) have been found around the gene of group II subfamily sPLA<SB>2</SB>. Subsequently, it has been found that the SNP10 (NCBI reference: rs584367) is a sensitive SNP relating to the reduction of BMI of COPD patient by using a genom DNA collected from 276 male COPD patients. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene associated with weight loss in chronic obstructive pulmonary disease (hereinafter sometimes referred to as COPD), and a polymorphism associated with weight loss in COPD, which is present in the gene or a DNA adjacent to the gene. About. The present invention also relates to a method for determining whether or not body weight is reduced in COPD using the polymorphism, that is, whether or not there is a poor prognosis associated with weight loss.

  Chronic obstructive pulmonary disease occupies a high position among 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 expected to rise to 5th by 2020 (non-patented) References 1, 2). This fatal disease is characterized by severe chronic inflammation occurring not only in the lungs but also systemically (Non-patent Documents 3 to 7). In particular, chronic and systemic inflammatory responses have an important impact on survival and prognosis. It has been reported that unexplained weight loss (muscle loss, muscle weakness, and adipose tissue depletion), which is a feature of advanced COPD, is associated with systemic inflammation (Non-Patent Documents 3 to 11). . This unexplained weight loss is a clinically significant problem because it is the most important factor that limits physical performance, impairs quality of life, and leads to poor prognosis, regardless of the severity of airflow obstruction (Non-Patent Documents 12 and 13).

  The main cause of the presence of chronic and systemic inflammation in COPD patients has not yet been elucidated. Recent studies have demonstrated a relationship between systemic metabolic abnormalities in COPD patients and elevated levels of pro-inflammatory mediators such as tumor necrosis factor (TNF) -α, a multifunctional cytokine in the circulating blood (Non-Patent Documents 3 to 7). TNF-α (cachexin), like other cachexia patients with chronic wasting disease, is associated with accelerated metabolism and protein turnover, leading to skeletal muscle protein loss and adipose tissue wasting ( Non-patent documents 3 to 7, 14). However, just as chronic smoking is a necessary but not sufficient factor to cause COPD, not all COPD patients show weight loss during the course of disease (Non-Patent Document 15). In fact, the prevalence of unexplained weight loss is particularly high in patients with severe COPD, approximately 50% of these patients, but to approximately 10-15% in patients with mild to moderate COPD It can be seen (Non-Patent Document 15). Similarly, the systemic inflammation profile believed to be related to the basic mechanism of weight loss is different for each individual with COPD, and the level of pro-inflammatory mediators in circulating blood in patients with COPD Is observed in a wide distribution, which is an interesting result (Non-Patent Documents 3 to 11). Weight loss is directly related to disease severity (Non-patent document 15) and phenotype (Non-patent document 16), and it is a genetic factor (smoking susceptibility) that COPD occurs only in some smokers. As explained by the presence of the gene), this important COPD prognostic determinant is the possibility that unexplained weight loss is also explained by genetic factors (Non-Patent Documents 17 and 18), The present inventors examined. The genes that may be involved in the weight loss process have not been elucidated so far.

Phospholipase A ₂ (hereinafter also referred to as PLA ₂ ) is an enzyme responsible for the mobilization of fatty acids including arachidonic acid (hereinafter also referred to as AA) from phospholipids (Non-patent Document 19). PLA ₂ is classified into high molecular weight cytoplasmic PLA ₂ (hereinafter sometimes referred to as cPLA ₂ ) and low molecular weight secretory PLA ₂ (hereinafter sometimes referred to as sPLA ₂ ) (Non-patent Document 19). ). In patients with systemic inflammatory diseases such as septic shock and extensive burns (Non-Patent Documents 20, 21) and autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease (Non-Patent Documents 22 and 23). in the plasma, it has been reported that a large amount of sPLA ₂ is released. Initially, these extracellular enzymes were thought to play an important role in inflammation by releasing AA that could be later converted to pro-inflammatory prostaglandins and leukotrienes. However, recent studies suggest that the pro-inflammatory effect of sPLA ₂ is not limited to the initiation of AA metabolism. Rather, some diseases associated with high levels of extracellular sPLA ₂ are characterized by a significant increase in plasma or tissue concentrations of pro-inflammatory cytokines such as TNF-α and IL-1β (non- Patent Document 24). Furthermore, sPLA ₂ has been reported to induce degranulation and production of pro-inflammatory cytokines from various cells involved in inflammatory and immune responses by an autocrine mechanism (Non-patent Document 19, 24). These effects are exerted by a mechanism independent of enzyme activity, and are mediated by interaction with a membrane receptor specific to sPLA ₂ or non-specific (Non-patent Document 24). Thus, proinflammatory cytokines and sPLA ₂ are thought to enhance each other's synthesis, thereby forming an amplification circuit for the propagation of inflammatory responses.

To date, 10 sPLA ₂ isoforms (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals (Non-patent Document 25). These isoforms, except for group III isoforms, have a highly conserved catalytic site, a Ca ²⁺ binding loop, and a common molecular weight of 14-19 kDa (Non-patent Document 25). Of these sPLA ₂ isoforms, sPLA ₂ -IIA, sPLA ₂ -IIC, sPLA ₂ -IID, sPLA ₂ -IIE, sPLA ₂ -IIF, and sPLA ₂ -V have the same chromosomal locus (1p34-p36 ), Which are often referred to as group II subfamily sPLA ₂ (Non-patent Document 25). The biological feature of the group II subfamily sPLA ₂ is that almost all isoforms, except sPLA ₂ -IIC (a pseudogene in humans), are associated with inflammatory and immune processes (non-patented) Reference 25).

The effect of sPLA ₂ pro-inflammatory properties on weight loss in COPD patients has not been investigated.

Prior art document information related to the invention of the present application is shown below.
Murray CJL, et al. Science. 1996; 274: 740-743. Pauwels RA, et al. Am J Respir Crit Care Med. 2001; 163: 1256-1276. Oudijk EJ, et al. Eur Respir J. 2003; 46: 5S-13S. Gan WQ, et al. Thorax. 2004; 59: 574-580. Andreassen H, et al. Eur Respir J. 2003; 46: 2S-4S. Wouters EF, et al. Chest. 2002; 121: 127S-130S. Agusti AG, et al. Eur Respir J. 2003; 21: 347-360. Vernooy JH, et al. Am J Respir Crit Care Med. 2002; 166: 1218-1224. 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. Schols AM, et al. Am J Respir Crit Care Med. 1998; 157: 1791-1797. Landbo C, et al. Am J Respir Crit Care Med. 1999; 160: 1856-1861. Argiles JM, et al. FASEB J. 1997; 11: 743-751. Schols AM, et al. Am Rev Respir Dis. 1993; 147: 1151-1156. Engelen MP, et al. Clin Nutr. 1999; 18: 275-280. Silverman EK, et al. Chest. 2000; 117: 273S-274S. Silverman EK, et al. Am J Respir Crit Care Med. 1998; 157: 1770-1778. Touqui L, et al. Curr Mol Med. 2001; 1: 739-754. Sorensen J, et al. Intensive Care Med. 1994; 20: 555-561. Nakae H, et al. Burns. 1995; 21: 422-426. Lin MK, et al. J Rheumatol. 1996; 23: 1162-1166. Haapamaki MM, et al. Scand J Clin Lab Invest. 1999; 59: 279-287. Granata F, et al. Int Arch Allergy Immunol. 2003; 131: 153-163. Murakami M, et al. J Biochem (Tokyo). 2002; 131: 285-292.

The present invention has been made in view of such a situation, and the object thereof is related to a gene related to weight loss in COPD, and weight loss in COPD existing in the gene or a DNA region adjacent to the gene. Is to find polymorphisms.
It is another object of the present invention to provide a method for predicting and determining whether body weight is reduced in COPD, using as an index a polymorphism related to weight loss in COPD existing in the gene or a DNA region adjacent to the gene.

In order to solve the above problems, the inventors focused on the pro-inflammatory properties of sPLA ₂ with respect to chronic and systemic inflammation, which is a potential cause of weight loss in COPD patients. Then, by searching the nucleic acid database, 12 single nucleotide polymorphisms (SNPs) were found around the genes of the group II subfamily sPLA ₂ .

Next, by using genomic DNA from 276 male COPD patients, we lost weight in COPD patients due to the conversion of group II subfamily sPLA ₂ genes resulting from these SNPs It was verified whether or not. Fisher's direct method, logistic regression analysis, and ANCOVA all showed statistical significance between SNP10 (NCBI SNP reference: rs584367, SEQ ID NO: 1 at position 10001) and decreased BMI in COPD patients SNP10 was shown to be a sensitive SNP associated with decreased BMI in COPD patients. Further, SNP 10, since a mutation site that causes an amino acid change of sPLA ₂ -IID within the gene, sPLA ₂ -IID it was suggested a susceptibility gene that causes weight loss in patients with COPD.

That is, the present inventors have found that SNP10 is a sensitive SNP associated with a decrease in BMI in COPD patients, and that weight loss in COPD patients is due to polymorphisms in the sPLA ₂ -IID gene, The present invention has been completed.

More specifically, the present invention provides the following (1) to (19).
(1) A method for determining whether or not body weight is decreased in chronic obstructive pulmonary disease, comprising detecting a mutation in a PLA2G2D gene or a nearby DNA region of the gene in a subject.
(2) The method according to (1), wherein the weight loss leads to poor prognosis.
(3) The method according to (1) or (2), wherein the mutation causes a mutation in the amino acid sequence of the protein encoded by the gene.
(4) The method according to any one of (1) to (3), wherein the mutation is a single nucleotide polymorphism mutation.
(5) A method for determining whether or not body weight is reduced 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 PLA2G2D gene or a DNA region in the vicinity of the gene in a subject.
(B) A step of determining that the subject loses weight in chronic obstructive pulmonary disease when a mutation is detected in the base type of the polymorphic site determined in (a).
(6) The method according to (5), wherein a decrease in weight leads to a poor prognosis.
(7) The polymorphic site is a site on the PLA2G2D gene region, and is a polymorphic site at any of positions 7469, 10001, or 12420 in the base sequence described in SEQ ID NO: 1. (5) Or the method as described in (6).
(8) The method according to (5) or (6), wherein the polymorphic site is a site on the PLA2G2D gene region and is a polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1.
(9) From (5) to (8), the base species at position 10001 in the base sequence described in SEQ ID NO: 1 is mutated from C to T at the site on the PLA2G2D gene region. ) Any one of the methods.
(10) A method for determining whether or not body weight is reduced in chronic obstructive pulmonary disease for a subject, comprising the following steps (a) and (b).
(A) A step of detecting a protein mutation comprising the amino acid sequence set forth in SEQ ID NO: 2 in the subject.
(B) The step of determining that the subject loses weight in chronic obstructive pulmonary disease when a mutation is detected in the protein as a result of (a).
(11) The method according to claim 10, wherein the weight loss leads to poor prognosis.
(12) The method according to (10) or (11), wherein the mutation in the protein is a mutation involving amino acid substitution.
(13) The method according to any one of (10) to (12), wherein the mutation in the protein is a mutation of Gly at position 80 of the amino acid sequence shown in SEQ ID NO: 2 to Ser.
(14) The method according to any one of (1) to (13), wherein a biological sample derived from a subject is used as a test sample.
(15) To determine whether body weight is decreased in chronic obstructive pulmonary disease, which comprises an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to the DNA comprising the polymorphic site according to (7) Drugs.
(16) A drug for determining whether body weight decreases in chronic obstructive pulmonary disease, comprising a solid phase on which a nucleotide probe that hybridizes with the DNA containing the polymorphic site described in (7) is immobilized.
(17) A drug for determining whether body weight decreases in chronic obstructive pulmonary disease, comprising a primer oligonucleotide for amplifying the DNA containing the polymorphic site according to (7).
(18) A drug for determining whether body weight is decreased in chronic obstructive pulmonary disease, comprising an antibody that binds to the protein described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2.
(B) A protein in which Gly at position 80 of the amino acid sequence shown in SEQ ID NO: 2 is mutated to Ser.
(19) The drug according to any one of (15) to (18), wherein weight loss leads to poor prognosis.

  According to the present invention, whether or not weight is decreased in COPD by detecting a gene associated with weight loss in COPD and a mutation in the DNA region on or near the gene, that is, poor prognosis associated with weight loss. A method of determining is provided. By detecting a polymorphism on the gene of the present invention or in a DNA region in the vicinity of the gene, it is possible to predict whether the body weight will subsequently decrease in the COPD patient. The present invention makes it possible to establish a more efficient strategy (so-called tailor-made medicine) in the prevention and treatment of weight loss in COPD. Specifically, patients who are predicted to lose weight by diagnosing whether COPD loses weight, that is, patients who are predicted to have a poor prognosis due to weight loss, increase the frequency of testing or strengthen nutritional support therapy It can be helpful when deciding on a treatment policy, and it can also lead to the development of new therapeutic agents that prevent weight loss.

  The inventors have identified genes and polymorphisms associated with weight loss in COPD. In COPD patients in whom weight loss occurs, mutations are significantly found in these genes or in the DNA region adjacent to the genes. It is possible to determine whether weight is lost in COPD patients.

  In the present invention, “weight loss” can be expressed as an index of the distribution or change of the BMI value. When the BMI value decreases, it can be considered that the weight has decreased. In addition, even if the weight is reduced as a result of any of the following reasons: loss of muscle mass, muscle weakness, or adipose tissue depletion, it can be regarded as the same as the “weight loss” symptom of the present invention. I can do it.

  In addition, the “weight loss” of the present invention is most directly linked to a poor prognosis. In the present invention, “poor prognosis” finally refers to shortening of life expectancy. Of COPD, the group with weight loss (the group with decreased BMI) has a clearly worse prognosis than the COPD patient group without it. That is, the remaining life expectancy is short. Although it differs depending on the literature, there are reports that the relative risk factor is about 20 times (Schols AM, et al. Am J Respir Crit Care Med. 1998; 157: 1791-1797 .; Landbo C, et al. Am J Respir Crit Care Med. 1999; 160: 1856-1861.). The main causes include easy infectivity such as lower respiratory tract infection such as pneumonia, exacerbation of right heart failure, fatal arrhythmia due to hypoxia, etc., and exacerbation of respiratory failure itself. All these complications increase in frequency as weight loss is observed (BMI value decreases), resulting in an average compared to the group without weight loss (group without BMI value) Proven to significantly and significantly shorten life expectancy (Schols AM, et al. Am J Respir Crit Care Med. 1998; 157: 1791-1797 .; Landbo C, et al. Am J Respir Crit Care Med 1999; 160: 1856-1861.). That is, the phenotype of weight loss indicates that the complications and respiratory failure itself lead to poor prognosis.

The gene found by the inventors to be associated with weight loss in COPD is PLA2G2D within the sPLA ₂ -IID gene (GenBank accession number: NM — 012400).
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 detects whether or not weight is reduced in COPD, that is, associated with weight loss, characterized by detecting a mutation in the PLA2G2D gene or a nearby DNA region of the gene for a subject. A method for determining poor prognosis is provided.

  In the present invention, “determining whether or not weight is reduced in COPD” includes a test for determining whether or not there is a high or low possibility of weight loss in a subject COPD patient. In the method of the present invention, when a mutation is detected in the PLA2G2D gene or a nearby DNA region of the gene, it is determined that the subject loses weight or has a predisposition to lose weight in COPD. Alternatively, it is determined that it is not resistant to, or does not have a predisposition to, weight loss in COPD.

  On the other hand, when a mutation is not detected in the PLA2G2D gene or a DNA region near the gene, it is determined that the subject is resistant to, or has a predisposition to resistance to, weight loss in COPD. Alternatively, it is determined that COPD does not lose weight or has no predisposition to lose weight.

  According to the method of the present invention, even if a subject does not suffer from COPD, it is possible to determine whether the possibility of weight loss in COPD is high or low when suffering from COPD. In addition, in the case of a subject who already suffers from COPD, it can be judged whether the possibility of progression of weight loss is high or low, and it can be used to determine a treatment policy such as nutritional support therapy. it can.

  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 PLA2G2D 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 weight loss in COPD in the PLA2G2D gene of the present invention or a DNA region adjacent to the gene in a subject. Therefore, the presence or absence of mutation is used as an index for the PLA2G2D gene of the present invention or a polymorphic site in the vicinity of the gene (determining the base type) to examine whether or not weight is lost in COPD. 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 PLA2G2D gene and the DNA sequence in the vicinity of the gene is shown in SEQ ID NO: 1.
In a preferred embodiment of the present invention, it is determined whether or not weight loss occurs in COPD, characterized by detecting a polymorphic mutation (base species at the polymorphic site) in the PLA2G2D gene of the present invention or a DNA region adjacent to 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 relates to a method for examining whether or not the subject loses weight in COPD, characterized by determining the base type of the polymorphic site in the PLA2G2D gene of the present invention or a DNA region adjacent to the gene of the present invention. I will provide a.

  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 PLA2G2D gene of the present invention or a DNA region adjacent to the gene. Specifically, as a polymorphic site that can be used in the test method of whether or not weight loss in COPD of the present invention is present, it is a PLA2G2D gene or a site on a DNA region in the vicinity of the gene, and described in SEQ ID NO: 1. One of the polymorphic sites at positions 7469, 10001, and 12420 in the base sequence can be exemplified. (In the present specification, these polymorphic sites may be simply referred to as “polymorphic sites of the present invention”).

略語の定義：NCBI=National Center for Biotechnology Information;
Gly=グリシン；Ser=セリン；sPLA₂=分泌型フォスフォリパーゼA₂ The 12 polymorphisms in the sPLA ₂ gene tested in the present invention are shown in Table 1 below.

Abbreviation definition: NCBI = National Center for Biotechnology Information;
Gly = glycine; Ser = serine; sPLA ₂ = secretory phospholipase A ₂

  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.

  The base type of the polymorphic site shown in the above table may indicate a base type on the complementary strand side with respect to the sequence shown in the sequence table, 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 the other result whether the positive strand or the negative strand is examined in the test. Can be determined. As for the human genome sequence, the international human genome project build34, which is said to be almost final, has been announced, and the sequences described in this specification are based on the results of the human genome international project build34.

  The person skilled in the art usually uses 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 sequence before and after it. 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 examples described later, there are some which are tested using complementary strands for each sequence described in SEQ ID NO: 1.

  In the test method for determining whether or not the body weight is reduced in COPD of the present invention, the polymorphic site at position 10001 in the PLA2G2D gene or a region on the DNA region in the vicinity of the gene, in the nucleotide sequence set forth in SEQ ID NO: 1. It is preferable to determine the base type.

  Further, in a preferred embodiment of the present invention, the PLA2G2D gene of the present invention or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence described in SEQ ID NO: 1 is T (hetero and homo) Is determined to be weight loss in COPD. Whether COPD patients lose weight or not, that is, poor prognosis associated with weight loss can be determined.

  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. By determining the species, it is also possible to test whether weight is lost in COPD. That is, this “neighboring polymorphic site” (for example, the above table) is applied to a small group of humans including patients with COPD patients who have developed weight loss whose base type is the above-mentioned base type. 1) is determined in advance.

  Next, the base type in the subject is determined for this “neighboring polymorphic site”, and compared with the base type determined in advance, it is possible to examine whether or not the body weight is reduced in COPD. If the base type is the same as the predetermined base type, the subject is determined to lose weight in COPD. With the test method of the present invention, it is possible to determine whether or not weight is lost in a COPD patient, and it can be used for determination of treatment policy, determination of drug dosage, presence of nutritional supplement therapy, and the like.

  For a small population of humans including those who have lost weight in COPD whose base type of the polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1 on the PLA2G2D gene is T, for example, a nearby polymorphic site, The base type of the polymorphic site at position 12420 is determined. When the frequency of T in the person who developed weight loss in the above-mentioned COPD was higher than that in the person who did not lose weight in the above-mentioned COPD, the nucleotide type of this site was 12420th polymorphic site in the subject. When the base species is examined and the base species at this site is also T, the subject is determined to lose weight in COPD.

  As described above, according to the present invention, a region on a gene related to weight loss in COPD has been clarified, so that it is examined whether or not weight is reduced in the above-mentioned COPD without imposing an excessive burden on those skilled in the art. It 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. These 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.

[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 form a hairpin structure alone. 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, it hybridizes to a PLA2G2D gene or a site on the DNA region in the vicinity of the gene, which contains a polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1, and has at least 15 nucleotides An oligonucleotide having a chain length can be used as a reagent (test agent) for examining whether or not weight is lost in COPD. 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 PLA2G2D 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 PLA2G2D 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 a test method for determining whether or not weight is lost (has a predisposition to sensitivity) in the COPD of the present invention. The reagent of the present invention includes the primer and / or probe of the present invention. In the test of whether or not weight loss is caused in COPD, DNA containing a PLA2G2D gene or a site on the DNA region in the vicinity of the gene and containing a polymorphic site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 1 is amplified. Primers and / or probes are used.

  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 weight is lost in COPD, which comprises a solid phase on which a nucleotide probe that hybridizes with DNA containing the polymorphic site of the PLA2G2D gene of the present invention is immobilized. It is a reagent for.
These are used for examinations using the polymorphic site of the present invention as an index. These preparation methods are as described above.

  The present invention also uses the expression level of PLA2G2D gene in a subject (biological sample derived from the subject) as an index to determine whether or not COPD loses weight or has a predisposition to lose weight. It is also possible. That is, the present invention, when the expression level of PLA2G2D gene in the subject is reduced compared to the control, it is determined that the subject loses weight or has a predisposition to lose weight in COPD. Provided is a method for testing whether COPD is weight loss or predisposed to weight loss.

  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 PLA2G2D gene in the test sample can be appropriately performed by those skilled in the art using known techniques. The “control” usually refers to the expression level of PLA2G2D gene in a biological sample derived from a healthy person. The expression of PLA2G2D gene in the present invention means both expression of mRNA transcribed from PLA2G2D gene and expression of protein encoded by PLA2G2D gene.

The present invention relates to a method for determining whether or not body weight is decreased in chronic obstructive pulmonary disease, including the following steps (a) and (b).
(A) A step of detecting a protein mutation comprising the amino acid sequence set forth in SEQ ID NO: 2 in the subject.
(B) The step of determining that the subject loses weight in chronic obstructive pulmonary disease when a mutation is detected in the protein as a result of (a).

In the present invention, protein mutation can include mutation due to addition, deletion, modification, or substitution of amino acids, and preferably includes mutation due to amino acid substitution. Further, specific examples of the mutation of the protein of the present invention include a mutation of the amino acid at position 80 of the amino acid sequence shown in SEQ ID NO: 2, more preferably 80 of the amino acid sequence shown in SEQ ID NO: 2. Can be mentioned that Gly at the position is mutated to Ser.
Detection of amino acid sequence mutations can be performed by methods known to those skilled in the art, such as mass spectrometry, amino acid N-terminal analysis, and immunochemical methods using antibodies.

When detecting an amino acid mutation of a protein by an immunochemical method, an antibody that binds to the protein described in (a) or (b) below can be used.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2.
(B) A protein in which Gly at position 80 of the amino acid sequence shown in SEQ ID NO: 2 is mutated to Ser.
The antibody of the present invention is not particularly limited as long as it recognizes the protein, but is preferably an antibody that specifically recognizes the protein.

The antibody used for detecting the protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used. As the antibody recognizing the protein of the present invention, a known antibody can be used. Alternatively, an antibody can be prepared by a method known to those skilled in the art using the protein as an antigen.
Specifically, for example, it can be produced as follows.

  A serum is obtained by immunizing a small animal such as a rabbit with the recombinant protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with the protein or GST. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the protein or synthetic peptide, or the like. In the case of a monoclonal antibody, for example, the protein or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells, and the cells and mouse myeloma cells are isolated. Are fused using a reagent such as polyethylene glycol, and a clone that produces an antibody that binds to the protein is selected from fused cells (hybridomas) produced by the method. Next, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, the protein Or by purification using an affinity column coupled with a synthetic peptide.

  Moreover, if it is a polyclonal antibody, it can acquire as follows, for example. Using the protein or fragment thereof as a sensitizing antigen, immunizing it according to a normal immunization method, fusing the resulting immune cells with known parental cells by a normal cell fusion method, by a normal screening method, A monoclonal antibody-producing cell (hybridoma) is screened. The antigen can be prepared according to a known method, for example, a method using baculovirus. When the immunogenicity of the antigen is low, immunization may be performed by binding to an immunogenic macromolecule such as albumin. Thereafter, the cDNA of the variable region (V region) of the antibody is synthesized from the hybridoma mRNA using reverse transcriptase, and the sequence of the obtained cDNA may be decoded by a known method.

  The antibody that recognizes the protein is not particularly limited as long as it binds to the protein, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, a human antibody, and the like can be appropriately used. In addition, recombinant antibodies that have been artificially modified for the purpose of reducing the heterologous antigenicity to humans, such as chimeric antibodies and humanized antibodies, can also be used. These modified antibodies can be produced using known methods. The chimeric antibody is a mammal other than a human, for example, an antibody comprising a heavy chain of a mouse antibody, a variable region of a light chain and a heavy chain of a human antibody, a constant region of a light chain, and the like, and encodes a variable region of a mouse antibody. It can be obtained by ligating DNA with DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host and producing it.

  A humanized antibody is also called a reshaped human antibody, and is a non-human mammal, for example, a complementarity determining region (CDR) of a mouse antibody transplanted to the complementarity determining region of a human antibody. There are also known general genetic recombination techniques. Specifically, several oligos prepared from DNA sequences designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody so as to have a portion overlapping the terminal portion. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with DNA encoding a human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it. As the FR of the human antibody to be ligated via CDR, one in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site.

  A method for obtaining a human antibody is also known. For example, human lymphocytes are sensitized with a desired antigen or a cell that expresses the desired antigen in vitro, and the sensitized lymphocyte is fused with a human myeloma cell such as U266 to have a desired human antibody having an antigen-binding activity. (See Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen. Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector having the sequence can be prepared and a human antibody can be obtained.

  The antibody used in the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field. The “antibody” in the present invention includes these conjugated antibodies.

  Preferred antibodies in the present invention include low molecular weight antibodies. The low molecular weight antibody is not particularly limited as long as it includes an antibody fragment lacking a part of a full-length antibody (whole antibody such as whole IgG) and has an ability to bind to an antigen. The antibody fragment of the present invention is not particularly limited as long as it is a part of a full-length antibody, but preferably contains a heavy chain variable region (VH) or a light chain variable region (VL), particularly preferably VH and VL. A fragment containing both. Specific examples of the antibody fragment include Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), and the like. In order to obtain such an antibody fragment, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors. Thereafter, it may be expressed in an appropriate host cell.

  The antibody of the present invention can be used as a drug for determining whether body weight decreases in chronic obstructive pulmonary disease. In the above drugs, in addition to the active ingredient antibody, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protein stabilizer (BSA, gelatin, etc.), preservative Etc. may be mixed as needed. A protein chip or the like pasted on the basis of the antibody of the present invention can also be used to determine whether or not weight is reduced in chronic obstructive pulmonary disease.

  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 2003-2004, 276 male patients whose genomic DNA was available after informed consent for 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. The patient was clinically stable for at least 3 months and had no signs of worsening clinical symptoms. 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.

<Pulmonary function test>
FVC and FEV _1.0 were measured with a standard spirometer (CHESTAC-25 Part II EX; Chest Co., Ltd., Tokyo, Japan). 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>
The group II subfamily sPLA ₂ genes and SNPs used in the study are shown in Table 1. Moreover, those chromosome positions are illustrated in FIG. Group II subfamily sPLA ₂ SNPs are dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), JSNP (http://snp.ims.u-tokyo.ac.jp), and Extracted from the Applied Biosystems genotyping Database (http://myscience.appliedbiosystems.com/genotype/). A total of 12 SNPs were derived from 5 genes sPLA ₂ -IIE, sPLA ₂ -IIA, sPLA ₂ -V, sPLA ₂ -IID, and sPLA ₂ -IIF, and the extent of weight loss in COPD for all SNPs The relationship with was tested. Genotyping was performed by fluorescent polymerase chain reaction. The allele and genotype frequencies of the tested SNPs were determined and combined with clinical data to perform statistical analysis.

<Type determination 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). The association of genotypes with the extent of weight loss in COPD (decrease in BMI in patients with COPD) and other clinical parameters, Fisher's direct method, logistic regression analysis, and covariance analysis (ANCOVA) (Wildt, Albert R. and Olli T. Ahtola (1978). Analysis of covariance. Quantitative Applications in the Social Sciences series # 12. Thousand Oaks, CA: Sage Publications. Both dominant and recessive genetic models were applied to these statistical analyses. Because age, lung function index, and smoking index are potential confounding variables for COPD patients and multivariate analysis needs to compensate for the effects of these confounding variables, logistic regression analysis and ANCOVA , Smoking index, and lung function index (forced expiratory volume per second-percentage of expected value). In particular, ANCOVA is used when the dependent variable is a numerical variable (BMI in the present invention), the independent variable is categorical and numerical (SNP genotype in the present invention), and there are several confounding variables. Can be applied. However, if there is any interaction between the independent variable (SNP genotype) and the confounding variable, ANCOVA cannot be applied. In the present invention, it was confirmed that there was no significant interaction between the SNP genotype and the confounding variable (interaction p-value> 0.05). To compare the clinical values of three different genotype groups, a one-factor analysis of variance (ANOVA) test was used with a post hoc correction (Fisher's protected test). Results were expressed as mean ± SD and p <0.05 was considered significant. All these data analyzes were performed by SPSS ver.12.0.1J (SPSS Inc., Chicago, IL, 2003).

Example 1 Fisher's Direct Method and Logistic Regression Analysis The purpose of the present invention may be related to individual susceptibility to weight loss (clinical prominent and most important feature suggesting poor prognosis) in COPD patients To identify and identify genetic polymorphisms within the SNP of the group II subfamily sPLA ₂ invented by the inventors.
Therefore, the present inventor first has 100 people with the lowest BMI [phenotype A group: BMI (kg / m ² ); 17.13 ± 1.29 (12.49-18.98), age (years); 73.8 ± 7.1, smoking index; 1117 ± 573, FEV _1.0 (% of expected value); 40.18 ± 20.37; n = 100] and conversely 100 people with the highest BMI [Phenotype a group: BMI (kg / m ² ); 23.83 ± 1.98 ( 21.63 to 32.74), age (years); 73.4 ± 6.7, smoking index; 1089 ± 551, FEV _1.0 (% of expected value); 53.62 ± 23.80; n = 100]. Fisher's direct method [Odds ratio (OR) (95% CI): 2.36 (1.34 to 4.18), p = 0.004] and logistic regression [OR: 2.10 (1.13 to 3.90), p = 0.919] are inferior models Showed that SNP10 (NCBI SNP reference: rs584367) within sPLA ₂ -IID was significantly associated with the degree of weight loss in COPD patients (Table 2: in each SNP tested in the present invention) Fischer's direct method and logistic regression analysis are shown). Analysis shows that the SNP10 mutant allele (thymine: T) in the sPLA ₂ -IID gene (SEQ ID NO: 1) is more than double the weight loss in COPD than the non-mutant allele (cytosine: C) (phenotype a group). It was also shown to increase the risk of (phenotype A group) (p <0.05). Furthermore, this nucleic acid change (C → T) also caused an amino acid modification (glycine: Gly → serine: Ser) (Gly80Ser) of the sPLA ₂ -IID protein (Table 1). Logistic regression analysis, pulmonary function index assessed by (a percentage of the expected value) FEV _1.0 is a significant risk factor in the phenotype group A, (ratio expected (%)) FEV _1.0 1 % Reduction also showed a 1.03-fold increased risk of COPD weight loss (phenotype A group) (p <0.001, data not shown). The test value of Hardy-Weinberg equilibrium of SNP10 was 0.497, and the p value of Chi-square test was 0.481. The allele frequency of SNP-10-C was 0.6825, and the allele frequency of SNP-10-T was 0.3175. SNP on sPLA2-IID was located in the vicinity of SNP10, and SNP11 showing strong linkage disequilibrium showed a significant difference, but other SNPs showed no significant difference.
This suggests that SNP10 is a sensitive SNP associated with a decrease in BMI in COPD patients.

略語の定義：OR=オッズ比; CI=信頼区間（confidence interval）
フィッシャーの直説法；フィッシャーの直接法を用いた表現型A群と表現型a群のSNP遺伝子型の頻度の違いの比較
ロジスティック回帰分析；年齢、喫煙指数、肺機能指数（FEV_1.0（期待値に対する割合（％））についてのロジスティック回帰分析
すべての解析データはSPSS ver.12.0.1J (SPSS Inc., シカゴ. IL, 2003)を用いて得た。

Abbreviation definition: OR = odds ratio; CI = confidence interval
Fisher's direct method; comparison of SNP genotype frequency differences between Phenotype A and Phenotype a using Fisher's direct method Logistic regression analysis; Age, smoking index, lung function index (FEV _1.0 (with respect to expected value) Logistic regression analysis for percentage (%) All analysis data were obtained using SPSS ver.12.0.1J (SPSS Inc., Chicago. IL, 2003).

[Example 2] Results of genotype characterization of ANCOVA and SNP Next, in order to confirm the results of Fisher's direct method and logistic regression analysis, all COPD patients (n = 276) were analyzed. The ANCOVA test also showed a significant difference between the BMI value (logarithmic transformation) of the mutation group (CT + TT) and the BMI value of the non-mutation group (CC) in the recessive model of SNP10 [mean (SE): 1.293 (0.005) ) Vs. 1.317 (0.006), p = 0.003, Table 3: T test for log BMI on each SNP tested in this study, and ANCOVA for log BMI]. All registered COPD patients were stratified into four classes of commonly used BMI values (Schols AM, et al. Am J Respir Crit Care Med. 1998; 157: 1791-1797.). In addition, BMI values were corrected using ANCOVA and stratified into 6 classes. Those with a T allele that is a mutant allele (CT + TT) have a BMI value (Figure 2a) (Fischer's direct method: p = 0.00329) and an ANCOVA corrected BMI value (Figure 2b) (Fischer's direct method: In p = 2.33x10 ^-17 ), the distribution was significantly different from that without CC (CC). The association between this phenotype (weight loss in COPD) and the gene polymorphism resulted in those with a T-allele that was mutated in a lower BMI value layer (FIGS. 2a and 2b). The BMI mean value of the mutation “Ser” group (Gly / Ser + Ser / Ser: n = 148) was 1.2 (kg / m ² ) lower than the other non-mutation group (Gly / Gly: n = 128) (p = 0.0005) FIG. 3a). The significant difference in BMI adjusted by ANCOVA between the two groups (Gly / Ser + Ser / Ser: n = 143, Gly / Gly: n = 118) showed a more pronounced p-value (p <0.0001, FIG. 3b) . Other diagnostic parameters for the severity of COPD by three genotypes of SNP10 within the sPLA ₂ -IID (CC: Gly / Gly, CT: Gly / Ser, and TT: Ser / Ser) are summarized in Table 4. ing. There were no significant differences in the levels of these parameters, including lung function, between the three groups. The mean BMI of the heterozygous combination “Gly / Ser” and the BMI adjusted by ANCOVA were lower than that of the homozygous combination “Ser / Ser”. These results may have been caused by the small number of homozygous samples and their relatively large deviations in the analysis. These results are also illustrated in FIGS. 4a and 4b, respectively.

In addition, SNP10 adjacent SNPs (SNP11) did not reach statistical significance for the most part, but showed low p-values in the recessive model, so sPLA ₂ -IID is a cause of weight loss in COPD patients It is also suggested that it is a gene.

log BMに対するt検定；Tテストによる2つの群のLog BMIの平均値の比較
log BMIに対するANCOVA；年齢、喫煙指数、肺機能指数（FEV_1.0（期待値に対する割合 （％））についてのANCOVA
すべての解析データはSPSS ver.12.0.1J (SPSS Inc., シカゴ. IL, 2003)を用いて得た。

t test for log BM; comparison of the average values of Log BMI of two groups by T test
log ANCOVA for BMI; ANCOVA for age, smoking index, lung function index (FEV _1.0 (% of expected value))
All analysis data were obtained using SPSS ver.12.0.1J (SPSS Inc., Chicago. IL, 2003).

The sPLA ₂ -IID protein consists of 125 amino acids followed by a 20-residue prepeptide (M _r = 14,500, SEQ ID NO: 2), and sPLA with respect to the number and position of cysteine residues and overall identity (48%) _It is most similar to _2- IIA (Ishizaki J, et al. J Biol Chem. 1999; 274: 24973-24979.). sPLA ₂ -IID is the constitutive expression in immune organs and digestive organs of the human, mouse lung, thymus, and upregulated by systemic proinflammatory stimulation in some restricted tissue such as spleen Suggests a functional role in the progression of the inflammatory process (Ishizaki J, et al. J Biol Chem. 1999; 274: 24973-24979., Murakami M, et al. Eur J Biochem. 2002; 269 : 2698-2707.). In fact, the search for nucleic acid databases, consistent with inducible properties related to proinflammatory signaling, binding motifs of the TATA box and AP-1 and NF-[kappa] B in the 5 'flanking promoter region of human sPLA ₂ -IID gene The existence of The SNP form of SNP10 (NCBI SNP reference: rs584367) is a missense mutation that causes an amino acid change (Gly80Ser) in the sPLA ₂ -IID protein. The position of the amino acid polymorphism is not in Ca ²⁺ binding loop in the catalytic domain that is highly conserved in Group II subfamily sPLA _2, it is completely conserved in Group II subfamily sPLA ₂ Located next to the cysteine residue (Ishizaki J, et al. J Biol Chem. 1999; 274: 24973-24979., Suzuki N, et al. J Biol Chem. 2000; 275: 5785-5793.). Thus, it is believed that single amino acid changes result in structural modifications of the sPLA ₂ -IID protein, and as a result affect the functional properties of this molecule to some extent.

Based on the results of the present invention, the pathophysiological relationship between weight loss and sPLA ₂ -IID protein amino acid polymorphism (SNP10) in COPD patients will be discussed. First, the ability to degranulate or synthesize pro-inflammatory cytokines by specific or promiscuous membrane receptors of inflammatory cells is modified or enhanced by amino acid changes (Gly → Ser) in the sPLA ₂ -IID protein There seems to be a possibility. Persistent and systemic hyperinflammatory conditions result in weight loss in COPD patients with heterozygous combination “Gly / Ser” or homozygous combination “Ser / Ser” (Table 4: COPD with SNP10 genotype) Patient characteristics and FIG. 4). Second, one possible function of the sPLA ₂ -IID protein is the digestion of phospholipids in nutrients (Ishizaki J, et al. J Biol Chem. 1999; 274: 24973-24979.). A functional change in this property of the sPLA ₂ -IID protein due to the amino acid polymorphism (SNP10) is thought to affect the efficiency of phospholipid digestion in COPD patients. That is, COPD patients with genotype heterozygous combination “Gly / Ser” or homozygous combination “Ser / Ser” show lower phospholipid digestion efficiency, resulting in weight loss despite nutritional supplement therapy (Table 4 and FIG. 4).

略語の定義：Gly=グリシン；Ser=セリン；BMI=肥満度指数（Body Mass Index）；FVC=強制肺活量（forced vital capacity）；FEV_1.0=1秒間努力呼気容量；BW(kg)：p<0.05, BMI（kg/m²）：p<0.01, ANCOVAによる調整されたBMI（kg/m²）：年齢、喫煙指数、肺機能指数（FEV_1.0（期待値に対する割合（％））についてのANCOVA；値は平均値±SD 、p<0.0001 一元配置分散分析；Gly/Gly vs. Gly/SerおよびGly/Gly vs. Ser/Ser：フィッシャーのＰＬＳＤ法（Fisher's protected least significant difference test）；動脈PO₂ (torr)および動脈PCO₂（torr）：相当数の患者が採血中に酸素補給を受けた試験。

Definition of abbreviations: Gly = glycine; Ser = serine; BMI = Body Mass Index; FVC = forced vital capacity; FEV _1.0 = 1 second forced expiratory capacity; BW (kg): p <0.05 , BMI (kg / m ² ): p <0.01, BMI adjusted by ANCOVA (kg / m ² ): Age, smoking index, lung function index (FEV _1.0 (% of expected value) ANCOVA; Values are mean ± SD, p <0.0001 one-way analysis of variance; Gly / Gly vs. Gly / Ser and Gly / Gly vs. Ser / Ser: Fisher ’s protected least significant difference test (PLSD); arterial PO ₂ ( torr) and arterial PCO ₂ (torr): Trials in which a significant number of patients received supplemental oxygen during blood collection.

Is a diagram showing the gene and SNP chromosomal location of the group II subfamily sPLA ₂ used in the present invention. Group II subfamily sPLA ₂ forms a cluster at the same chromosomal locus (1p34-p36), and the 12 SNPs used in the present invention are dbSNP (http: //www.ncbi.nlm.nih .gov / SNP /), JSNP (http://snp.ims.u-tokyo.ac.jp), and Applied Biosisytems genotyping Database (http: //myscience.appliedbiosystems) Extracted from .com / genotype /). It is a figure which shows distribution of BMI value of all the registration COPD patients. All registered COPD patients are stratified into four commonly used BMI values (Schols AM, et al. Am J Respir Crit Care Med. 1998; 157: 1791-1797.), And ANCOVA is used to calculate BMI values. Corrections were also made to stratify into 6 classes. Those with a T allele that is a mutant allele (CT + TT) have a BMI value (Figure 2a) (Fischer's direct method: p = 0.00329) and an ANCOVA corrected BMI value (Figure 2b) (Fischer's direct method: In p = 2.33x10 ^-17 ), the distribution was significantly different from that without CC (CC). In the ANCOVA test, 15 patients were excluded from the analysis due to lack of accurate information on smoking history. FIG. 3a shows the BMI values of the “Ser” group (Gly / Ser + Ser / Ser: n = 148) and the other non-mutated group (Gly / Gly: n = 128) according to the SNP10 genotype in sPLA ₂ -IID (kg / m ² ) [average (95% CI)]. This figure proved the significant difference between the mutation groups (Student t test; p = 0.0005). FIG. 3b shows the value of BMI adjusted by ANCOVA for these two groups (Gly / Ser + Ser / Ser: n = 143, Gly / Gly: n = 118) according to the genotype of SNP10 in sPLA ₂ -IID [average ( 95% CI)]. An ANCOVA test demonstrated even more significant significance (p <0.0001). In the ANCOVA test, 15 patients were excluded from the analysis due to lack of accurate information on smoking history. FIG. 4a is a diagram showing box plots of BMI values (kg / m ² ) for three genotypes of SNP10 in sPLA ₂ -IID. This figure proved a significant difference between the three groups (ANOVA test using Fisher's protected test as a post-hoc correction; p <0.01, also described in Table 4). FIG. 4 b is a diagram showing a box plot of BMI values (kg / m ² ) adjusted by ANCOVA according to three genotypes of SNP10 in sPLA ₂ -IID. A more significant difference between the three groups has been demonstrated (p <0.0001, also explained in Table 4). In the ANCOVA test, 15 patients were excluded from the analysis due to lack of accurate information on smoking history. The skip value was deleted from the figure.

Claims (19)

  A method for determining whether or not body weight is decreased in chronic obstructive pulmonary disease, comprising detecting a mutation in a PLA2G2D gene or a nearby DNA region of the gene for a subject.   The method according to claim 1, wherein the weight loss leads to poor prognosis.   The method according to claim 1 or 2, wherein the mutation causes a mutation in the amino acid sequence of the protein encoded by the gene.   The method according to any one of claims 1 to 3, wherein the mutation is a single nucleotide polymorphism mutation. A method for determining whether or not body weight is reduced 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 PLA2G2D gene or a DNA region in the vicinity of the gene in a subject.
(B) A step of determining that the subject loses weight in chronic obstructive pulmonary disease when a mutation is detected in the base type of the polymorphic site determined in (a).   6. The method according to claim 5, wherein the weight loss leads to a poor prognosis.   The polymorphic site is a site on the PLA2G2D gene region, and is a polymorphic site at any of positions 7469, 10001, or 12420 in the base sequence described in SEQ ID NO: 1. The method described.   The method according to claim 5 or 6, wherein the polymorphic site is a site on the PLA2G2D gene region and is a polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1.   9. The base species according to claim 5, wherein the base species at position 10001 in the base sequence described in SEQ ID NO: 1 is mutated from C to T at a site on the PLA2G2D gene region. The method described. A method for determining whether or not body weight is reduced in chronic obstructive pulmonary disease for a subject, comprising the following steps (a) and (b):
(A) A step of detecting a protein mutation comprising the amino acid sequence set forth in SEQ ID NO: 2 in the subject.
(B) The step of determining that the subject loses weight in chronic obstructive pulmonary disease when a mutation is detected in the protein as a result of (a).   The method according to claim 10, wherein weight loss leads to poor prognosis.   The method according to claim 10 or 11, wherein the mutation in the protein is a mutation involving amino acid substitution.   The method according to any one of claims 10 to 12, wherein the mutation in the protein is a mutation in Gly at position 80 of the amino acid sequence shown in SEQ ID NO: 2 to Ser.   The method according to any one of claims 1 to 13, wherein a biological sample derived from a subject is subjected to a test as a test sample.   A drug for determining whether body weight decreases 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 whether body weight is reduced 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 whether body weight decreases in chronic obstructive pulmonary disease, comprising a primer oligonucleotide for amplifying DNA comprising the polymorphic site according to claim 7. An agent for determining whether or not weight is decreased in chronic obstructive pulmonary disease, comprising an antibody that binds to the protein described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2.
(B) A protein in which Gly at position 80 of the amino acid sequence shown in SEQ ID NO: 2 is mutated to Ser.   The drug according to any one of claims 15 to 18, wherein a decrease in body weight leads to a poor prognosis.

Images