JP2006314315A - Method for examining pulmonary function and abnormality and composition therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating and diagnosing the tendency and/or degree of COPD (chronic obstructive pulmonary disease) and pulmonary emphysema in smokers or non-smokers by utilizing analytical results of changed gene expression and hereditary polymorphisms, and to provide a method for diagnosing the tendency and/or degree of pulmonary dysfunction related to the COPD and/or emphysema. <P>SOLUTION: The method comprises a method for analyzing one or more polymorphisms selected from specific substances. In the method, the genotypes exhibit onset predispositions or potential onset risks of the COPD and/or pulmonary emphysema possessed by the object. The polymorphisms are directly analyzed or polymorphisms in linkage disequilibrium with the polymosphisms are analyzed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for examining lung function and / or abnormality using genetic polymorphism and altered gene expression in smokers and non-smokers, and in particular, chronic obstructive pulmonary disease (COPD). The present invention relates to a method for diagnosing the predisposition and / or extent of emphysema. The invention also relates to a method of diagnosing the predisposition and / or extent of pulmonary dysfunction associated with COPD and / or emphysema.

  Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in developed countries and is a leading cause of readmission in hospitals worldwide. COPD is characterized by insidious inflammation and progressive lung destruction. COPD is clinically manifested only when smokers suffering from COPD experience severe dyspnea when more than 50% of lung function has already been irreversibly lost. This loss of pulmonary function is clinically detected by a decrease in the expiratory flow rate (particularly the forced expiratory volume per second, or FEV1). Over 95% of COPD results from smoking, but only about 20% of smokers go to COPD (sensitive smokers). Studies have surprisingly found that only about 16% of lung loss is accounted for by smoking. A study comparing the concordance of siblings (twin and non-twins) for a large number of families revealed a strong familial tendency, so the search for a COPD disease susceptibility gene (or a gene that changes this disease) is ongoing. It has been.

  Despite advances in the treatment of airway diseases, current therapies cannot significantly change the natural progression of COPD from respiratory failure to death with progressive lung function loss. Smoking cessation reduces this decrease in lung function, but if sensitive smokers do not quit within about 20 years of smoking, loss of lung function is significant and symptoms of worsening dyspnea may be reversed. Can not. Smoking cessation studies show that ways to help smokers quit are not always successful. Similar to the discovery of the relationship between serum cholesterol and coronary artery disease, a better understanding of the factors involved in COPD will enable the development of tests to identify at-risk smokers and the harmful effects of smoking. There is a need to be able to find new therapies to reduce the effect.

  Many epidemiological studies have shown that the distribution of lung function has three peaks when smoking over 20 boxes x years. In other words, there is a certain percentage of smokers (resistant smokers) who maintain normal lung function even over 60 boxes x years, and the lung function gradually decreases but never shows symptoms There is a certain percentage of people, and there is a certain percentage of people who are inevitably losing lung function and become COPD. This means that there are three types of smokers: those who are resistant to COPD, those who are moderately risky, and those who are higher risk (named sensitive smokers). Show.

  COPD is a diverse disease that includes varying degrees of emphysema and chronic bronchitis that develop as part of the regeneration process that occurs after chronic exposure to tobacco smoke and other inflammatory trauma from air pollution. Is included. Many genes appear to be involved in the development of COPD.

  A number of biomarkers have been identified so far to help diagnose and assess the propensity to develop various lung diseases. Among them, as disclosed in PCT International Application PCT / NZ02 / 00106 (published as WO 02/099134, which is incorporated in its entirety), for example: There are single nucleotide polymorphisms such as: A-82G in the promoter of the gene encoding human macrophage elastase (MMP12); T in codon 10 of the gene encoding transforming growth factor β (TGFβ) → C; C + 760G of the gene encoding superoxide dismutase 3 (SOD3); T-1296C in the promoter of the gene encoding metalloproteinase tissue inhibitor 3 (TIMP3); A polymorph that is in equilibrium.

  A further assessment that allows the subject to assess the predisposition to developing lung disease (eg, chronic obstructive pulmonary disease (COPD) or emphysema), or the predisposition to developing COPD / pulmonary dysfunction associated with emphysema, especially if the subject is a smoker Would be desirable and useful.

  The object of the present invention is mainly such a biomarker and a method for evaluating the predisposition to the disease and / or the risk of developing the disease and / or the onset using the biomarker.

  The present invention is primarily based on the fact that certain polymorphisms are more frequently seen in subjects suffering from COPD and / or emphysema than in control subjects. Analysis of these polymorphisms reveals a relationship between the genotype and the predisposition and / or potential risk of developing COPD and / or emphysema.

Thus, according to one aspect of the invention, a method for determining whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or COPD and / or A method for diagnosing the possibility of developing emphysema,
-765 C / G in the promoter of the gene encoding cyclooxygenase 2 (COX2);
105 C / A within the gene encoding interleukin 18 (IL18);
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G in the promoter of the gene encoding plasminogen activator inhibitor 1 (PAI-1);
874 A / T in the gene encoding interferon gamma (IFN-γ);
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14);
Exon 1 +49 C / T in the gene encoding elafin;
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding matrix metalloproteinase 1 (MMP1);
Including analyzing one or more polymorphisms selected from the group consisting of:
Methods are provided in which the genotype indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of onset.

  It is possible to analyze these polymorphisms directly or to analyze polymorphisms that are in linkage disequilibrium with these polymorphisms.

  Linkage disequilibrium is a phenomenon in genetics in which two or more mutations or polymorphisms are inherited together because they are so close together. This means that when determining the genotype, if the presence of one polymorphism is found, the other is also presumed. (Reich D.E. et al., “Linkage disequilibrium in the human genome”, Nature, 2001, Vol. 411, pp. 199-204.)

In addition to one or more of the above polymorphisms,
16 arginine / glycine in the gene encoding β2 adrenergic receptor (ADBR);
130 arginine / glutamine (G / A) within the gene encoding interleukin 13 (IL13);
298 aspartate / glutamate (T / G) in the gene encoding NO synthase 3 (NOS3);
Isoleucine 105 valine (A / G) in the gene encoding glutathione S-transferase P (GST-P);
Glutamic acid 416 aspartic acid (T / G) in the gene encoding vitamin D binding protein (VDBP);
Lysine 420 threonine (A / C) in the gene encoding VDBP;
-1055 C / T within the promoter of the gene encoding IL13;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G in the promoter of the gene encoding interleukin 1B (IL1B);
Tyrosine 113 histidine T / C in the gene encoding microsomal epoxide hydrolase (MEH);
Arginine 139 G / A in the gene encoding MEH;
Glutamine 27 glutamic acid C / G in the gene encoding ADBR;
Analysis of one or more polymorphisms selected from the group consisting of can be included.

  It is possible to analyze these polymorphisms directly or to analyze polymorphisms that are in linkage disequilibrium with these polymorphisms.

As polymorphisms that can be combined and analyzed,
-1607 1G / 2G (for the 2G allele only) within the promoter of the gene encoding metalloproteinase 1 (MMP1);
-1562 C / T in the promoter of the gene encoding metalloproteinase 9 (MMP9);
M1 (GSTM1) null in the gene encoding glutathione S transferase 1 (GST-1);
1237 G / A in the 3 ′ region of the gene encoding α1-antitrypsin;
-82 A / G within the promoter of the gene encoding MMP12;
T → C within codon 10 of the gene encoding TGFβ;
760 C / G within the gene encoding SOD3;
-1296 T / C within the promoter of the gene encoding TIMP3;
an S mutation in the gene encoding α1-antitrypsin;
There is one or more polymorphisms selected from the group consisting of

  Here again, the method of the present invention can analyze polymorphisms that are in linkage disequilibrium with the polymorphs specifically mentioned.

  Thus, in another particular embodiment, the above method comprises a -765 C / G polymorphism within the promoter of a gene encoding COX2, and / or a polymorphism in linkage disequilibrium with this polymorphism. This genotype indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the -765 CC genotype or -765 CG genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In yet another specific embodiment, the above method comprises analyzing a 105 C / A polymorphism within the gene encoding IL18 and / or a polymorphism in linkage disequilibrium with this polymorphism. At least, this genotype indicates the subject's predisposition to COPD and / or emphysema and / or the risk of potential development.

  In this embodiment, the 105 AA genotype preferably exhibits an increased predisposition to and / or an increased risk of developing COPD and / or emphysema.

  In yet another particular embodiment, the above method comprises a -133 G / C polymorphism within the promoter of a gene encoding IL18 and / or a polymorphism in linkage disequilibrium with this polymorphism. This genotype indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, it is preferred that the -133 CC genotype is indicative of a predisposition to COPD and / or emphysema and / or an increased risk of developing it.

  In yet another particular embodiment, the above method is -675 4G / 5G polymorphism within the promoter of the gene encoding PAI-1 and / or linkage disequilibrium with this polymorphism It includes at least a polymorphic analysis, which indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the -675 5G5G genotype preferably exhibits an increased predisposition to COPD and / or emphysema and / or an increased risk of developing.

  In yet another specific embodiment, the above method comprises 874 A / T polymorphism in the gene encoding IFN-γ and / or a polymorphism in linkage disequilibrium with this polymorphism. Including at least analysis, this genotype indicates the predisposition and / or potential risk of developing COPD and / or emphysema in the subject.

  In this embodiment, it is preferred that the 874 AA genotype indicates a predisposition to COPD and / or emphysema and / or an increased risk of potential development.

  In yet another particular embodiment, the above method results in a +489 G / A polymorphism in the gene encoding tumor necrosis factor alpha (TNFα) and / or linkage disequilibrium with this polymorphism. The genotype indicates the predisposition and / or potential risk of developing COPD and / or emphysema in the subject.

  In this embodiment, the +489 AA genotype or +489 AG genotype is preferably indicative of a predisposition to COPD and / or emphysema and / or an increased risk of potential onset.

  In this embodiment, the +489 GG genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In yet another specific embodiment, the above method comprises analyzing a C89Y A / G polymorphism in the gene encoding SMAD3 and / or a polymorphism that is in linkage disequilibrium with this polymorphism. At least, this genotype indicates the subject's predisposition to COPD and / or emphysema and / or the risk of potential development.

  In this embodiment, the C89Y AA genotype or AG genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In this embodiment, the C89Y GG genotype preferably exhibits an increased predisposition to COPD and / or emphysema and / or an increased risk of developing.

  In yet another particular embodiment, the method described above results in an E469K A / G polymorphism within the gene encoding intercellular adhesion molecule 1 (ICAM1) and / or linkage disequilibrium with this polymorphism. The genotype indicates the predisposition and / or potential risk of developing COPD and / or emphysema in the subject.

  In this embodiment, the E469K GG genotype preferably exhibits an increased predisposition to and / or an increased risk of developing COPD and / or emphysema.

  In yet another specific embodiment, the method is glycine 881 arginine G / C polymorphism in the gene encoding caspase (NOD2) and / or linkage disequilibrium with this polymorphism. It includes at least a polymorphic analysis, which indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the glycine 881 arginine GC or CC genotype is preferably indicative of a predisposition to COPD and / or emphysema and / or an increased risk of developing it.

  In yet another particular embodiment, the above method results in a 161 G / A polymorphism in the gene encoding mannose-binding lectin 2 (MBL2), and / or in linkage disequilibrium with this polymorphism. Analysis of at least one polymorphism, which indicates the subject's predisposition to COPD and / or emphysema and / or the potential risk.

  In this embodiment, the 161 GG genotype preferably exhibits a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In yet another specific embodiment, the method is -1903 G / A polymorphism within the gene encoding chymase 1 (CMA1) and / or linkage disequilibrium with this polymorphism. It includes at least a polymorphic analysis, which indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the -1903 AA genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In yet another specific embodiment, the above method comprises arginine 197 glutamine G / A polymorphism in the gene encoding N-acetyltransferase 2 (NAT2), and / or linkage disequilibrium with this polymorphism. Analysis of at least one polymorphism, which indicates the subject's predisposition to COPD and / or pulmonary emphysema and / or potential risk.

  In this embodiment, the arginine 197 glutamine AA genotype preferably exhibits a predisposition to COPD and / or emphysema and / or a reduced risk of potential development.

  In yet another particular embodiment, the method described above results in a -366 G / A polymorphism within the gene encoding 5 lipo-oxygenase (ALOX5) and / or linkage disequilibrium with this polymorphism. The genotype indicates the predisposition and / or potential risk of developing COPD and / or emphysema in the subject.

  In this embodiment, the -366 AA genotype or -366 AG genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In this embodiment, the -366 GG genotype preferably exhibits an increased predisposition to and / or an increased risk of developing COPD and / or emphysema.

  In yet another specific embodiment, the above method is in linkage disequilibrium with and / or a HOM T2437C polymorphism within the gene encoding heat shock protein 70 (HSP70). It includes at least a polymorphic analysis, which indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the HOM T2437C CC genotype or CT genotype preferably exhibits an increased predisposition to COPD and / or emphysema and / or an increased risk of developing.

  In this embodiment, it is preferred that the HOM T2437C TT genotype indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential development.

  In yet another specific embodiment, the above method comprises the +13924 T / A polymorphism in the gene encoding calcium-dependent chloride channel 1 (CLCA1) and / or a linkage not linked to this polymorphism. It includes at least an analysis of balanced polymorphisms, which indicates the subject's predisposition to COPD and / or emphysema and / or the risk of potential development.

  In this embodiment, the +13924 AA genotype preferably indicates an increased predisposition to COPD and / or emphysema and / or an increased risk of developing.

  In yet another specific embodiment, the above method comprises: -159 C / T polymorphism in the gene encoding monocyte differentiation antigen CD-14 (CD-14), and / or It includes at least an analysis of polymorphisms that are in linkage disequilibrium, and this genotype indicates the subject's predisposition to COPD and / or emphysema and / or the potential risk.

  In this embodiment, the -159 CC genotype preferably exhibits an increased predisposition to COPD and / or emphysema and / or an increased risk of developing it.

  In yet another particular embodiment, the above method comprises exon 1 +49 C / T polymorphism in the gene encoding elafin and / or a polymorphism in linkage disequilibrium with this polymorphism. This genotype indicates the subject's predisposition to COPD and / or emphysema and / or potential risk of developing.

  In this embodiment, the exon 1 +49 CT genotype or TT genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  In yet another particular embodiment, the above method comprises the -1607 1G / 2G polymorphism within the promoter of the gene encoding MMP1 (only for the 1G allele) and / or no linkage with this polymorphism. It includes at least an analysis of balanced polymorphisms, which indicates the subject's predisposition to COPD and / or emphysema and / or the risk of potential development.

  In this embodiment, the 1G1G genotype or 1G2G genotype preferably indicates a predisposition to COPD and / or emphysema and / or a reduced risk of potential onset.

  The polymorphism in each of the above specific embodiments can be analyzed alone or in any combination of two or more, the latter being more preferred.

In yet another embodiment, the above method further comprises an operation of analyzing one or more of the polymorphisms in combination with at least one other polymorphism, wherein the at least one polymorphism The choice is
16 arginine / glycine in the gene encoding ADBR;
298 aspartic acid / glutamic acid (T / G) within the gene encoding NOS3;
Lysine 420 threonine (A / C) in the gene encoding VDBP;
Glutamic acid 416 aspartic acid (T / G) in the gene encoding VDBP;
Isoleucine 105 valine (A / G) in the gene encoding GST-P;
874 A / T in the gene encoding IFN-γ;
130 arginine / glutamine (G / A) within the gene encoding IL13;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G within the promoter of the gene encoding IL1B;
Tyrosine 113 histidine T / C in the gene encoding MEH;
Arginine 139 G / A in the gene encoding MEH;
Glutamine 27 glutamic acid C / G in the gene encoding ADBR;
-1055 C / T within the promoter of the gene encoding IL13;
-1607 1G / 2G within the promoter of the gene encoding MMP1;
-1562 C / T in the promoter of the gene encoding MMP9;
M1 null in the gene encoding GST-1;
1237 G / A in the 3 ′ region of the gene encoding α1-antitrypsin;
-82 A / G within the promoter of the gene encoding MMP12;
T → C within codon 10 of the gene encoding TGFβ;
760 C / G within the gene encoding SOD3;
-1296 T / C within the promoter of the gene encoding TIMP3;
an S mutation in the gene encoding α1-antitrypsin;
Polymorphisms that are in linkage disequilibrium with these;
From a group of

  These methods are particularly effective for smokers (both current and former smokers).

  In the context of the present invention, the term “polymorphism” describes all variants and mutations, including total or partial absence of genes (eg, null mutations). It will be understood that it is used. Similarly, in the context of the present invention, the term “single nucleotide polymorphism” or “SNP” includes single nucleotide nucleotide substitutions and polymorphisms due to short deletions or insertions.

  Furthermore, it will be appreciated that the polymorphisms described in this specification fall into two categories. That is, a polymorphism ("protective polymorphism" or "protective SNP") that shows that the analysis indicates a predisposition to COPD and / or emphysema and / or reduced potential risk ) And polymorphisms ("susceptibility polymorphism" or "susceptibility SNP") that can be found by analysis to indicate an increased predisposition to COPD and / or emphysema and / or an increased risk of developing Is possible).

Thus, the present invention provides a method for determining whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or the likelihood of developing COPD and / or emphysema. A method of diagnosing,
Examining the presence or absence of at least one protective SNP allele associated with a reduced tendency for COPD and / or emphysema, and / or a reduced potential risk, and / or a reduced incidence;
In the absence of any protective SNP allele, the presence or absence of at least one sensitive SNP allele associated with increased tendency for COPD and / or emphysema and / or potential risk and / or increased incidence Includes the operation of examining
The presence of one or more protective SNP alleles indicates a reduced tendency for COPD and / or emphysema and / or a reduced potential risk and / or a reduced incidence, and which protective SNP allele The absence of the gene, combined with the presence of at least one susceptible SNP allele, increases the tendency for COPD and / or emphysema and / or increases the potential risk and / or increases the incidence. There is further provided a method indicating:

Selection of the at least one protective SNP allele is
A -765 C allele within the promoter of the gene encoding COX2;
130 arginine / glutamine A alleles within the gene encoding IL13;
298 aspartate / glutamate T allele within the gene encoding NOS3;
Lysine 420 threonine A allele within the gene encoding VDBP;
Glutamic acid 416 aspartic acid T allele within the gene encoding VDBP;
An isoleucine 105 valine A allele within the gene encoding GSTP-1;
an S allele within the gene encoding α1-antitrypsin;
A +489 G allele within the gene encoding TNFα;
A -308 G allele within the gene encoding TNFα;
A C89Y A allele within the gene encoding SMAD3;
A 161 G allele within the gene encoding MBL2;
A -1903 A allele within the gene encoding CMA1;
Arginine 197 Glutamine A allele within the gene encoding NAT2;
Histidine 139 arginine G allele within the gene encoding MEH;
A -366 A allele within the gene encoding ALOX5;
A HOM 2437 T allele within the gene encoding HSP70;
Exon 1 +49 T allele within the gene encoding elafin;
Glutamine 27 glutamate G allele within the gene encoding ADBR; and
A -1607 1G allele within the gene encoding MMP1;
It is preferable to carry out from the group consisting of

In yet another aspect, the present invention provides a method for determining whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or COPD and / or emphysema. A method for diagnosing the possibility of the onset of
Examining the presence or absence of at least one protective SNP genotype associated with a reduced tendency for COPD and / or emphysema, and / or a reduced potential risk, and / or a reduced incidence;
In the absence of any protective SNP genotype, examine the presence or absence of at least one susceptible SNP genotype associated with COPD and / or emphysema trends and / or potential risk and / or development Including operations;
Further provided are methods wherein the genotype indicates a predisposition to COPD and / or emphysema, and / or a potential risk and / or likelihood of onset.

In one embodiment, selecting the at least one SNP genotype comprises:
-765 CC genotype or CG genotype within the promoter of the gene encoding COX2;
130 arginine / glutamine AA genotype within the gene encoding IL13;
298 aspartate / glutamate TT genotype within the gene encoding NOS3;
Lysine 420 threonine AA genotype or AC genotype within the gene encoding VDBP;
Glutamic acid 416 aspartic acid TT genotype or TG genotype within the gene encoding VDBP;
Isoleucine 105 valine AA genotype within the gene encoding GSTP-1;
MS genotype within the gene encoding α1-antitrypsin;
+489 GG genotype within the gene encoding TNFα;
-308 GG genotype within the gene encoding TNFα;
C89Y AA or AG genotype within the gene encoding SMAD3;
161 GG genotype within the gene encoding MBL2;
-1903 AA genotype within the gene encoding CMA1;
Arginine 117 glutamine AA genotype within the gene encoding NAT2;
Histidine 139 arginine GG genotype within the gene encoding MEH;
-366 AA or AG genotype within the gene encoding ALOX5;
HOM T2437C TT genotype within the gene encoding HSP70;
Exon 1 +49 CT genotype or TT genotype within the gene encoding elafin; and
Glutamine 27 glutamic acid GG genotype within the gene encoding ADBR; and
-1607 1G1G genotype or 1G2G genotype within the gene encoding MMP1;
From a group of

In yet another embodiment, the above method comprises:
+ 760GG genotype or + 760CG genotype within the gene encoding SOD;
-1296TT genotype within the promoter of the gene encoding TIMP3; and
CC genotype (homo P allele) within codon 10 of the gene encoding TGFβ;
An additional step of revealing the presence or absence of at least one protective SNP genotype selected from the group consisting of:

In yet another embodiment, the predisposition to COPD and / or emphysema, and / or the potential risk of development, and / or selection of the at least one susceptible SNP genotype associated with the development,
105 AA genotype within the gene encoding IL18;
-133 CC genotype within the promoter of the gene encoding IL18;
-675 5G5G genotype within the promoter of the gene encoding PAI-1;
-1055 TT genotype within the promoter of the gene encoding IL13;
874 TT genotype within the gene encoding IFN-γ;
+489 AA genotype or AG genotype within the gene encoding TNFα;
-308 AA genotype or AG genotype within the gene encoding TNFα;
C89Y GG genotype within the gene encoding SMAD3;
An E469K GG genotype within the gene encoding ICAM1;
Glycine 881 arginine GC genotype or CC genotype within the gene encoding NOD2;
-511 GG genotype within the gene encoding IL1B;
Tyrosine 113 histidine TT genotype within the gene encoding MEH;
-366 GG genotype within the gene encoding ALOX5;
HOM T2437C CC genotype or CT genotype within the gene encoding HSP70;
+13924 AA genotype within the gene encoding CLCA1; and
-159 CC genotype within the gene encoding CD-14;
From a group of

In yet another embodiment, the predisposition to COPD and / or emphysema, and / or the potential risk of development, and / or selection of the at least one susceptible SNP genotype associated with the development,
-82 AA genotype within the promoter of the gene encoding MMP12;
-1562 CT genotype or -1562 TT genotype within the promoter of the gene encoding MMP9;
1237 AG genotype or 1237 AA genotype (allelic genotype Tt or tt) within the 3 ′ region of the gene encoding α1-antitrypsin; and
2G2G genotype within the promoter of the gene encoding MMP1;
From a group of

  In yet another aspect, the present invention determines whether a subject has a predisposition to and / or potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema and / or COPD and / or emphysema. In which the -765 C allele is present in the promoter of the gene encoding COX2 and / or the S allele is present in the gene encoding α1-antitrypsin. The presence or absence of one or more alleles is associated with a reduced predisposition to and / or potential risk of developing COPD and / or emphysema, and / or COPD And / or methods showing reduced likelihood of developing emphysema are provided.

  In yet another aspect, the present invention determines whether a subject has a predisposition to and / or potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema and / or COPD and / or emphysema. Of the gene encoding COX2 within the promoter of -765 CC genotype or CG genotype and / or the gene encoding α1-antitrypsin Reducing the predisposition and / or potential risk of developing COPD and / or emphysema, including the operation of determining whether the type is present or absent. And / or a method showing a reduced likelihood of developing COPD and / or emphysema is provided.

In one particularly preferred embodiment of the invention, a method for determining whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema,
-765 C / G in the promoter of the gene encoding COX2;
105 C / A within the gene encoding IL18;
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G within the promoter of the gene encoding PAI-1;
874 A / T in the gene encoding IFN-γ;
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14);
Exon 1 +49 C / T in the gene encoding elafin; and
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding MMP1;
In addition to one or more polymorphisms selected from the group consisting of
16 arginine / glycine in the gene encoding ADBR;
130 arginine / glutamine (G / A) within the gene encoding IL13;
298 aspartic acid / glutamic acid (T / G) within the gene encoding NOS3;
Isoleucine 105 valine (A / G) in the gene encoding GSTP;
Glutamic acid 416 aspartic acid (T / G) in the gene encoding VDBP;
Lysine 420 threonine (A / C) in the gene encoding VDBP;
-1055 C / T within the promoter of the gene encoding IL13;
S mutation of the gene encoding α1-antitrypsin;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G in the promoter of the gene encoding interleukin 1B (IL1B);
Tyrosine 113 histidine T / C in the gene encoding microsomal epoxide hydrolase (MEH);
Arginine 139 G / A within the gene encoding MEH; and
Glutamine 27 glutamic acid C / G in the gene encoding ADBR;
A method is provided that includes analyzing one or more polymorphisms selected from the group consisting of:

  In one preferred embodiment of the invention, the presence of two or more protective genotypes indicates a reduced risk of developing COPD and / or emphysema.

  In yet another preferred embodiment of the invention, the presence of two or more susceptible genotypes indicates an increased risk of developing COPD and / or emphysema.

  In yet another preferred embodiment of the invention, the presence of two or more protective genotypes, regardless of the presence or absence of one or more susceptible genotypes, may indicate the onset of COPD and / or emphysema Indicates that the risk is decreasing.

Yet another feature is a method for determining whether a subject has a predisposition to and / or potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or the likelihood of developing COPD and / or emphysema A method for diagnosing
-765 C / G in the promoter of the gene encoding cyclooxygenase (COX2);
105 C / A within the gene encoding interleukin 18 (IL18);
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G in the promoter of the gene encoding plasminogen activator inhibitor 1 (PAI-1);
874 A / T in the gene encoding interferon-γ (IFN-γ);
16 arginine / glycine in the gene encoding the β2 adrenergic receptor;
130 arginine / glutamine (G / A) within the gene encoding interleukin 13 (IL13);
298 aspartic acid / glutamic acid in the gene encoding NO synthase 3 (NOS3);
Isoleucine 105 valine (A / G) in the gene encoding glutathione S-transferase P (GST-P);
Glutamic acid 416 aspartic acid (T / G) in the gene encoding vitamin D binding protein (VDBP);
Lysine 420 threonine (A / C) in the gene encoding vitamin D binding protein;
-1055 C / T in the promoter of the gene encoding interleukin-13;
an S mutation in the gene encoding α1-antitrypsin;
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14);
Exon 1 +49 C / T in the gene encoding elafin;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G in the promoter of the gene encoding interleukin 1B (IL1B);
Tyrosine 113 histidine T / C in the gene encoding microsomal epoxide hydrolase (MEH);
Arginine 139 G / A in the gene encoding MEH;
Glutamine 27 glutamate C / G within the gene encoding ADBR; and
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding MMP1;
A method is provided that includes analyzing two or more polymorphisms selected from the group consisting of:

  In various embodiments, any one or more of the above methods includes analyzing an amino acid present at a position that maps to codon 298 of the gene encoding NOS3.

  Preferably, the presence of glutamic acid at said position indicates a predisposition and / or potential risk of developing COPD and / or emphysema, and / or the likelihood of developing COPD and / or emphysema.

  The presence of asparagine at the location preferably indicates a reduced risk of developing COPD and / or emphysema.

  In various embodiments, any one or more of the methods described above includes analyzing an amino acid present at a position that maps to codon 420 of a gene encoding a vitamin D binding protein.

  Preferably, the presence of threonine at said position indicates a predisposition and / or potential risk of developing COPD and / or emphysema, and / or the likelihood of developing COPD and / or emphysema.

  Preferably the presence of lysine at said position indicates a reduced risk of developing COPD and / or emphysema.

  In various embodiments, any one or more of the methods described above comprises analyzing an amino acid present at a position that maps to codon 89 of the gene encoding SMAD3.

  In various embodiments, any one or more of the above methods includes analyzing an amino acid present at a position that maps to codon 469 of the gene encoding ICAM1.

  In various embodiments, any one or more of the methods described above comprises analyzing the amino acid present at a position that maps to codon 881 of the gene encoding NOD2.

  In various embodiments, any one or more of the above methods includes analyzing an amino acid present at a position that maps to codon 197 of the gene encoding NAT2.

  In various embodiments, any one or more of the above methods includes analyzing an amino acid present at a position that maps to codon 113 of the gene encoding MEH.

  In various embodiments, any one or more of the above methods includes analyzing an amino acid present at a position that maps to codon 139 of the gene encoding MEH.

  In various embodiments, any one or more of the methods described above comprises analyzing an amino acid present at a position that maps to codon 27 of a gene encoding ADBR.

  In another aspect, the present invention provides a group of nucleotide probes and / or nucleotide primers for use in the preferred methods of the invention described herein. Preferably, the nucleotide probe and / or nucleotide primer covers the polymorphic region of the gene or can be used to cover the polymorphic region of the gene using the nucleotide probe and / or nucleotide primer. .

  In yet another aspect, the present invention provides one or more susceptible polymorphisms or protective polymorphisms described herein for use in the prognosis prediction and / or diagnosis and / or screening described herein. There is provided a nucleic acid microarray having a substrate on which a nucleic acid sequence capable of hybridization with a nucleic acid sequence encoding a nucleic acid sequence or a sequence complementary thereto is displayed.

  As another aspect, the present invention, when used in conjunction with the susceptibility or protection polymorphisms described herein, for use in the prognosis prediction and / or diagnosis and / or screening described herein. An antibody microarray is provided that includes a substrate that displays antibodies capable of binding to the product of a gene whose expression is up- or down-regulated.

  In yet another aspect, the present invention provides a method for treating a subject having an increased predisposition to COPD and / or emphysema and / or an increased risk of developing or developing COPD and / or emphysema. A method is provided that includes reproducing the presence and / or functioning effect of a protective genotype with respect to genotype or phenotype in the body of the subject.

  In yet another aspect, the present invention is a subject who has an increased predisposition to COPD and / or emphysema and / or an increased risk of developing or has developed COPD and / or emphysema Detectable upregulates or downregulates gene expression, such that the physiologically active concentration of the gene product is outside the normal range for the subject's age and gender A method of treating a subject having a different susceptibility polymorphism comprising the step of returning a physiologically active concentration of the expressed gene product to a range that is normal for the age and sex of the subject is provided. The

  In yet another aspect, the present invention is a subject having a predisposition to and / or potential risk of developing COPD and / or pulmonary emphysema, which is COPD and / or pulmonary emphysema and encodes COX2. A method of treating a subject whose GG genotype is known to exist at the position of the -765 C / G polymorphism present in the promoter of the gene, and reducing COX2 activity in the subject's body There is provided a method comprising the step of administering to the subject an agent capable of

  In one embodiment, the drug is a COX2 inhibitor, or a non-steroidal anti-inflammatory drug (NSAID), and the COX2 inhibitor is from the group consisting of Celebrex (celecoxib), Bextra (valdecoxib), Biox (rofecoxib) It is preferable to select.

  As yet another aspect of the present invention, a subject having a predisposition to and / or a potential risk of developing COPD and / or emphysema, wherein AA is located at the position of the 105 C / A polymorphism in the gene encoding IL18. A method is provided for treating a subject known to have a genotype, comprising administering to the subject an agent capable of increasing IL18 activity in the subject's body. .

  As yet another aspect of the present invention, a gene having a predisposition to COPD and / or emphysema and / or a potential risk of developing, or a subject having COPD and / or emphysema, and encoding IL18 A method of treating a subject whose CC genotype is known to be present at the position of -133 G / C polymorphism in the subject, and which can increase the activity of IL18 in the subject's body A method is provided that includes administering to the subject.

  In yet another aspect, the present invention relates to a subject having a predisposition to and / or a potential risk of developing COPD and / or emphysema, wherein the subject has COPD and / or emphysema and encodes PAI-1. A method of treating a subject known to have a 5G5G genotype at the position of the -675 4G / 5G polymorphism within the promoter of the gene that increases PAI-1 activity in the subject's body There is provided a method comprising the step of administering to the subject an agent capable of being administered.

  In yet another aspect, the present invention provides a subject with a predisposition to and / or a potential risk of developing COPD and / or emphysema, or a subject who has COPD and / or emphysema and encodes IFN-γ. A method of treating a subject whose AA genotype is known to be present at the position of the 874 A / T polymorphism within the gene, which may alter the activity of IFN-γ in the subject's body A method is provided that includes administering a possible agent to the subject.

  In yet another aspect, the present invention is a subject having a predisposition to and / or a potential risk of developing COPD and / or emphysema, which is subject to COPD and / or emphysema and encodes CD-14. A method of treating a subject whose CC genotype is known to be present at the position of the -159 C / T polymorphism within the gene, wherein the activity of CD-14 and / or IgE in the subject's body A method is provided that includes administering to the subject an agent capable of altering.

In yet another aspect, the present invention provides a method of screening for compounds that alter the expression and / or activity of a gene whose expression is upregulated or downregulated when associated with a susceptibility or defensive polymorphism, comprising:
Contacting the candidate compound with a cell containing a sensitive or protective polymorphism known to be associated with upregulation or downregulation of gene expression;
Measuring the expression of the gene after contact with the candidate compound,
Provided is a method wherein a change in expression level after the contacting step as compared to before the contacting step indicates the ability of the compound to alter the expression and / or activity of the gene.

  The cells are preferably human lung cells whose presence of the polymorphism has been confirmed by preliminary screening.

  The cell contains a sensitive polymorphism associated with upregulation of the gene expression, and the screening is to look for candidate compounds that downregulate the expression of the gene.

  Alternatively, the cell contains a sensitive polymorphism associated with downregulation of the expression of the gene, and the screening is to look for candidate compounds that upregulate the expression of the gene.

  In another embodiment, the cell contains a protective polymorphism associated with upregulation of the gene expression, and the screening is to look for candidate compounds that further upregulate expression of the gene.

  Alternatively, the cell contains a protective polymorphism associated with downregulation of the gene expression, and the screening is to look for candidate compounds that further downregulate the expression of the gene.

In another aspect, the present invention provides a method of screening for a compound that modulates the expression and / or activity of a gene whose expression is upregulated or downregulated when associated with a susceptible or protective polymorphism, comprising:
Candidate compounds in cells that contain a gene whose expression is up- or down-regulated when associated with a susceptibility or defense polymorphism, although the expression of that gene is not up- or down-regulated in that cell A contacting step;
Measuring the expression of the gene after contacting with the candidate compound,
Provided is a method wherein a change in expression level after the contacting step as compared to before the contacting step indicates the ability of the compound to alter the expression and / or activity of the gene.

  The cells are preferably human lung cells in which the presence of the gene and the baseline level of expression of the gene have been confirmed by preliminary screening.

  Gene expression is down-regulated when associated with a susceptible polymorphism, and the screening is preferably to look for candidate compounds that up-regulate the expression of that gene in the cell.

  Alternatively, gene expression is upregulated when associated with a susceptibility polymorphism, and the screening is to look for candidate compounds that downregulate the expression of that gene in the cell.

  In another embodiment, gene expression is upregulated when associated with a protective polymorphism and the screening is to look for candidate compounds that upregulate the expression of the gene in the cell.

  Alternatively, gene expression is down-regulated when associated with a defense polymorphism and the screening is to look for candidate compounds that down-regulate the expression of that gene in the cell.

  In yet another aspect, according to the present invention, a subject having a predisposition to COPD or emphysema, or a subject diagnosed with COPD or emphysema, can determine the physiologically active concentration of the expressed gene product for that subject's age and sex. A method of assessing the likelihood of responding to a prophylaxis or treatment that involves returning to the normal range, detecting the presence or absence of a susceptibility polymorphism in the subject's body The susceptibility polymorphism up-regulates or down-regulates the expression of the gene, so that the physiologically active concentration of the expressed gene product is outside the normal range for the age and sex of the subject. A method is provided that includes detecting the presence, and detecting the presence of the polymorphism indicates that the subject is likely to respond to the treatment.

  Several genetic variants (polymorphisms) of candidate genes were compared between smokers and blood donors in a case-control study. The majority of candidate genes have been confirmed (or appear to have been confirmed) to affect gene expression or protein function. Specifically, the frequency of polymorphisms was compared between control blood donors, resistant smokers, and smokers suffering from COPD (separated into early-onset and normal-onset). In the present invention, it is shown that both a protective polymorphism and a sensitive polymorphism derived from the polymorphism of the selected candidate gene exist.

  In one embodiment described herein, 17 susceptibility genetic polymorphisms and 19 protective genetic polymorphisms are identified. This is shown below.

  Susceptible genetic polymorphism is a polymorphism that, when present, indicates a predisposition to COPD and / or emphysema and / or an increased risk of onset, and / or the likelihood of developing COPD and / or emphysema. In contrast, protective genetic polymorphism, when present, indicates a reduced predisposition to and / or potential risk of developing COPD and / or emphysema and / or a reduced likelihood of developing COPD and / or emphysema It is polymorphic.

Many of the polymorphisms can be analyzed alone. It is the following:
-765 C / G in the promoter of the gene encoding COX2;
105 C / A within the gene encoding IL18;
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G within the promoter of the gene encoding PAI-1;
874 A / T in the gene encoding IFN-γ;
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14); and exon 1 +49 C / T in the gene encoding elafin;
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding MMP1;
A polymorphism in linkage disequilibrium with any of the above polymorphisms.

  These polymorphisms can be analyzed in combination of two or more, or other polymorphisms that indicate the predisposition and / or potential risk of developing COPD and / or emphysema in the patient (including Can be analyzed in combination with the remaining types).

  Specifically considered are combinations of the above polymorphisms and the polymorphisms described in PCT International Application PCT / NZ02 / 00106 (published as WO 02/099134).

  Assays involving polymorphic combinations are preferred.

  The statistical analysis, especially the statistical analysis of the combined effects of these polymorphisms, reveals the degree of risk for any smoker using the genetic assay of the present invention, and in particular, the risk of developing COPD. It can be seen that larger smokers can be identified. Such a combined analysis can be performed for a combination of only sensitive polymorphisms, for a combination of only protective polymorphisms, or for a combination of both. The analysis can also be performed in a stepped manner. That is, it is possible to first analyze the presence or absence of a protective polymorphism, and then analyze the sensitive polymorphism only where the protective polymorphism exists.

  Therefore, as described in this document, several proteins involved in the development of COPD by systematically analyzing the frequency of these polymorphisms in well-known smokers and non-smokers groups And, for prevention purposes, can improve the ability to identify which smokers are at greater risk of developing COPD-related lung dysfunction and COPD.

  Because only a small number of smokers suffer from one or a combination of these diseases, there is a “susceptible smoker” and the genetic mechanism and exposure to high levels of oxidants The risk seems to increase through the combined effect of.

  The method of the present invention is primarily concerned with the detection and identification of the above polymorphisms (all single nucleotide polymorphisms) associated with COPD. In general, single nucleotide polymorphisms (SNPs) are single base changes or point mutations that result in genetic changes from individual to individual. In the human genome, about one SNP occurs every 100 to 300 bases, and there is a possibility that it occurs in both the coding region and the non-coding region. Because of the duplication of the genetic code, the SNP in the coding region may or may not change the amino acid sequence of the protein produced. Non-coding SNPs can affect gene transcription, processing, and translation by altering gene expression, for example, by changing regulatory regions (eg, promoters, transcription factor binding sites, processing sites, ribosome binding sites) There is sex.

  SNP has recently attracted a great deal of attention in SNP discovery and detection as it facilitates large-scale association genetics studies. SNPs are very promising as markers for many phenotypic traits, including potential traits (eg, disease tendency and extent, wellness tendency, response to drugs (eg, sensitivity to drug side effects)). Findings about the relationship between a particular SNP and a phenotypic trait, combined with the knowledge about whether an individual has that particular SNP, can be used to better control the disease, control the goal of the disease, It will be possible to develop a better understanding and ultimately to easily find more effective treatments (eg, personalized treatment plans).

  In fact, many databases have been constructed for known SNPs, and for some such SNPs, many databases of biological effects related to SNPs have also been constructed. For example, NCBI's SNP database “dbSNP” is built into NCBI's Entrez system and can be searched in the same way as other Entrez databases (eg PubMed and GenBank). This database contains records of over 1.5 million SNPs mapped onto the human genome sequence. Input items for each dbSNP include the polymorphic sequence context (ie surrounding sequences), the frequency of polymorphisms (in the population or individuals), experimental methods, protocols, conditions for examining changes, etc. Information about the relationship between phenotypic traits and SNPs can also be included in the input.

At least one reason is that SNP has a potential impact on health and wellness, and so much effort has been invested in developing methods to identify SNPs reliably and quickly. Efforts are repeated. This is not an easy task. Part of the reason is that human genomic DNA is complex with a 3 × 10 ⁹ base-pair haploid genome, and appropriate sensitivity and discrimination conditions are required.

  One of the well-known methods in the prior art for determining genotypes for the purpose of SNP detection is DNA sequencing. This method requires allele-specific hybridization of the primer or probe; allele-specific incorporation of nucleotides (“one base extension” or “ Often referred to as “mini-sequencing”); allele-specific ligation (conjugation) of oligonucleotides (ligation chain reaction probe or ligation probe); allele-specific cleavage of oligonucleotides or PCR products by restriction enzymes (Restriction fragment length polymorphism analysis or RFLP); identification of allele-dependent differences in electrophoretic or chromatographic mobility; structure-specific invasive enzymes (eg, structure-specific enzymes).

  SNP can be directly determined and identified by DNA sequencing. For screening purposes, it is generally more specific to genome-wide sequencing (or even to targeting a portion of the genome) than to the advantage of specificity and accuracy The problem is bigger. At this stage, direct genome sequencing to detect SNPs is not cost effective and time consuming.

  Another method for analyzing SNPs is the so-called mini-sequencing method. This method involves the operation of hybridizing the primer to a DNA sequence adjacent to the SNP site on the test sample being examined. The primer is extended by one nucleotide using all four fluorescent dideoxynucleotides (A, C, G, T) with different tags and DNA polymerase. Only one of the four nucleotides (if homo) or two (if hetero) is incorporated. The incorporated base is complementary to the nucleotide at the SNP site.

  A number of methods have been developed to detect SNPs that rely on changes in the conformation of the nucleic acid.

  For example, single-stranded conformation polymorphisms (SSCP, Orita et al., PNAS, 1989, 86, 2766-2770) form single-stranded nucleic acids in solution under secondary conditions. It depends on ability. The secondary structure depends on the base composition and can be changed by single base substitution. Then, a difference occurs in the mobility of electrophoresis under non-denaturing conditions. Detection of various polymorphisms is generally done by radiography (when radiolabeled), by band silver staining, by hybridization with a probe fragment with a detectable label, or by using fluorescent PCR primers. Use and later detect the primer using, for example, an automated DNA sequencer.

  The tertiary structure of single-stranded DNA changes under various physical conditions (for example, temperature and ionic environment). Therefore, the sensitivity of SSCP depends on these and other conditions. Although some empirical rules appear when choosing conditions to isolate sequence variants in the context of a particular sequence, the prediction of whether a change can be detected under a given condition is particularly new This is not possible when in the context of an array. The ability of PCR-SSCP to detect changes is generally high and has been reported to be over 80% in a single trial for fragments less than 300 bp (Hayashi and Yandell, 1993). However, since the sensitivity is not 100%, it cannot be said that there is no change in the analyzed molecule just because there is no new band.

  Since the sensitivity of SSCP decreases as the fragment length increases, less than 300 bp is optimal. To detect changes in longer fragments (exons greater than 300 bp or the entire cDNA), use overlapping short primers or long PCR products digested with appropriate restriction enzymes prior to SSCP.

  Improvements in SSCP are well known in the prior art, for example using different gel running conditions (eg, at different temperatures, or using additives such as glycerol (5-10%) or sucrose (10%)) Use different gel matrices (eg small polyacrylamide crosslinker ratio (49: 1 = acrylamide: bis-acrylamide) (5-10% gel), GeneAmp, hydrolink gel, agarose gel) Or

  Other variations well known to those skilled in the art regarding SSCP include:

  RNA-SSCP. RNA base pairs are more stable than RNA-DNA base pairs, so single-stranded RNA should have more secondary repertoire than single-stranded DNA, and is expected to adopt more diverse conformations Is done. Thus, RNA-SSCP has the advantage of being able to analyze larger fragments because it is more sensitive to sequence changes. However, making RNA strands is generally inconvenient. This is because an additional step is generally required to perform transcription in vitro for RNA-SSCP. Moreover, although the transcripts are abundant, rSSCP can be detected with ethidium bromide, but this method has not been widely used because it is more complex and costly.

REF-SSCP. An improvement to SSCP, which incorporates restriction endonuclease fingerprinting and aims to reduce the size of large fragments to about 150 bp. After digestion, the product is mixed, labeled with ³² P at the end, denatured and electrophoresed under non-denaturing conditions. The information provided by this system contains two “components”. The gain or loss of restriction sites (restriction component information) and the abnormal mobility of fragments (“SSCP component”).

  ddF: dideoxy fingerprinting. Hybrid between dideoxy sequencing and SSCP. ddF includes an operation of performing non-denaturing electrophoresis after a Sanger sequencing reaction with one dideoxynucleotide terminator. The ddF pattern is divided into a “dideoxy component” and an “SSCP component”. The sensitivity of the dideoxy component is unchanged, but the SSCP component varies with gel conditions, temperature, segment size, and sequence relationships. This method has the advantage over standard SSCP in that it can determine a rough location that has changed.

  bi-ddF: Bidirectional dideoxy fingerprinting. This method can be regarded as a “second generation ddF” and performs a dideoxy-termination reaction simultaneously using two primers for each end. There are two advantages over ddF alone. The first is that most single base substitutions can be detected by the ddF component, and the second is that the SSCP component is increased because a change in mobility can be detected in the downstream or upstream direction. These advantages mean that bi-ddF can be used to screen larger regions of genomic DNA with greater sensitivity.

  Fluorescent PCR-SSCP. In this method, SSCP is adapted to an automated sequencer for detecting polymorphisms. For example, multiplex fluorescent PCR-SSCP is a method in which a PCR product is labeled with a multiplex fluorescent dye, digested with a restriction enzyme, SSCP is performed, and analysis is performed with an automated DNA sequencer that can detect the fluorescent dye. To detect fluorescent PCR-SSCP (CE-FSSCP), an electrophoresis technique based on an automated capillary using a molecular sieve polymer system has also been developed.

  Other methods using different mobility of different nucleic acid structures include denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). Electrophoresis of double-stranded DNA (dsDNA) is performed in a gradient in which the concentration of the denaturing agent (urea and / or formamide) gradually increases or in a gradient in which the temperature gradually increases. As the denaturant concentration increases or the temperature rises, each domain in the DNA dissociates according to the melting point (Tm) of the domain. When the strands dissociate, the mobility of electrophoresis decreases. If there is a 1 bp difference between two homoduplex DNAs, the Tm may differ by 1 ° C or more. When there is a base mismatch in the heteroduplex, there is a significant destabilization of the domain, resulting in a Tm difference of 6 ° C between the homoduplex and the heteroduplex. For these reasons, heteroduplexes between wild-type and mutant fragments are generally used for point change analysis. Radiolabels, ethidium bromide, and silver staining are commonly used for detection.

  This method can be used to analyze fragments up to about 1000 bp. Since the mobility shift decreases as the number of melting domains increases, the fraction of the detected change decreases as the fragment size increases.

  Heteroduplex analysis (HET) uses a similar molecular basis to identify SNPs. Heteroduplexes are produced by denaturing a mixture of wild-type and mutant DNA molecules with heat and then annealing again. In non-denaturing polyacrylamide gels, the mobility of electrophoresis differs between homoduplex and heteroduplex. These duplexes are generally detected by ethidium bromide or silver staining.

  Denaturing high-pressure liquid chromatography (HPLC) is another method for detecting SNPs using HPLC methods well known in the prior art instead of the above-described separation methods (eg gel electrophoresis). By detecting homoduplexes and heteroduplexes that elute at different rates, mismatched nucleotides and SNPs can be detected.

  Yet another method for detecting SNPs is based on the difference in sensitivity to cleavage with various reagents (eg, chemical cleavage agents, nucleolytic enzymes) between single-stranded and double-stranded nucleic acids. For example, under certain conditions, mismatches in RNA: DNA heteroduplexes are cleaved by RNase A. After cleavage, the labeled fragment is analyzed by DGGE. This method has several disadvantages. For example, this method is not suitable for analyzing a large number of SNPs because of the low efficiency of cleavage involving purine bases.

  Another method known as enzyme mismatch cleavage (EMC) involves incubating heteroduplexes resulting from heat denaturation of SNP-containing DNA or PCR products of wild-type and mutant alleles, respectively, for example bacteriophage T4 Cleave with endonuclease YII or T7 endonuclease I. The resulting fragment is analyzed by electrophoresis and detected by methods well known in the art.

  Similarly, in chemical cleavage (CCM), nucleotides that are mispaired within a heteroduplex are changed with chemicals commonly used in the Maxam-Gilbert sequencing method. Hydroxylamine reacts with mispaired cytosine residues, and osmium tetroxide reacts with mispaired thymidine residues. The chemically modified sites of DNA: DNA heteroduplex or DNA: RNA heteroduplex are then cleaved with piperidine to detect fragments (usually radioactively or fluorescently labeled).

  Yet another variation is known as cleavage fragment length polymorphism (CFLP). When a single strand of DNA is refolded after denaturation, a secondary structure is formed in a hairpin-like arrangement that is folded depending on the sequence. Cleavease I endonuclease cleaves the 5 'end of the hairpin loop at the junction of single and double stranded DNA. This produces a collection of fragments that are unique to that strand of DNA. Changes within the sequence (eg SNP) will change the secondary structure formed and the observed CFLP pattern. CFLP patterns are generally separated on short denaturing PAGE. Fragment detection is usually done by labeling one of the PCR primers.

  Mismatch binding proteins have also been used to detect changes in nucleic acid sequences. The change is detected, for example, by binding of MutS protein (a component of the E. coli DNA mismatch repair system) or human hMSH2 protein and GTBP protein to a double-stranded DNA molecule containing a mismatch base. These heteroduplexes are produced by a method well known in the prior art (for example, a method of denaturing by heat and then annealing again). The double stranded DNA is then incubated with the mismatch binding protein and changes are detected by mobility shift assay. For example, one simple assay is based on the fact that the heteroduplex is protected from exonuclease degradation by binding of the mismatch binding protein to the heteroduplex.

  Yet another example, the protein translation test (PTT), is based on a combination of PCR, transcription, and translation. This method selectively detects changes in translation termination. The change is revealed at the protein level by SDS-PAGE. A promoter (eg, T7 promoter) and a eukaryotic translation initiation sequence are ligated to the PCR primers. In the transcription-translation and subsequent reactions, the PCR product is used as a template in the presence of a detectable labeled amino acid. The size of the translation product is analyzed by gel electrophoresis. If a change results in a stop codon, translation is terminated prematurely, resulting in a protein product that is smaller than the original size. This assay is limited and can only detect changes that can be detected by different translation products. Therefore, no missense mutation is detected.

  Many methods currently used for the detection of SNPs include site-specific and / or allele-specific hybridization. These methods rely primarily on the oligonucleotides identifying and binding the target sequence containing the SNP of interest.

  The majority of methods for detecting or identifying SNPs by site-specific hybridization require that the target be amplified by methods such as PCR to improve sensitivity and specificity (eg, US Pat. No. 5,679,524, PCT Publication WO 98/59066, PCT publication WO 95/12607). When collecting whole human DNA, the target sequence including the SNP site is only a small part of the whole human DNA. For example, a 20 base pair target sequence is only 0.00000033% of the total DNA (in a normal diploid genome, there are two copies of a given target sequence, but each person may have a different SNP. The two copies can be considered different sites). Thus, a typical DNA sample of a few micrograms may be insufficient for many existing methods due to lack of sensitivity.

  More importantly, hybridization to a target sequence of 20 base pairs using an oligonucleotide short enough to distinguish one nucleotide, for example, does not necessarily hybridize only to the target region, but within the genome. It also binds to other areas in a small percentage. Since the amount of non-target DNA is overwhelmingly large, non-specific hybridization may generate a large background and a specific signal may be buried. Thus, amplifying one target region with PCR is a preferred step to dramatically reduce the amount of non-specific sequences. This amplification step is called “complexity reduction”. However, the fidelity of the PCR method is limited. Combining PCR primer pairs can produce spurious reaction products or lack specific regions. In addition, if untargeted sequences are copied or if an error is introduced into the target sequence, the number of errors contained in the final reaction product due to incorrect integration will result in 1 PCR amplification. It increases exponentially every time it is performed. Thus, PCR errors can be a major drawback when looking for rare mutations in a population of nucleic acids.

  In the method of Affymetrix (Santa Barbara, CA) and Nanogen (San Diego, CA), double-stranded DNA containing a single-base mismatch is more likely than a double-stranded base that is perfectly paired. Also take advantage of the fact that it is much more unstable. The presence of a matched duplex is detected by fluorescence.

  United States Application No. 20050059030 (which is incorporated herein in its entirety) selectively enriches the sequence of interest without pre-amplification or reduction of complexity, A method has been described for detecting single nucleotide polymorphisms contained in whole human DNA without the aid of any enzymatic reaction. This method utilizes a one-step hybridization that includes two hybridization events. Two hybridization events are an event in which the first portion of the target sequence hybridizes to the capture probe and an event in which the second portion of the target sequence hybridizes to the detection probe. Both hybridization events occur during the same reaction. The target sequence first binds to the capture oligonucleotide and then hybridizes to the detection probe (eg, nanoparticle). Alternatively, the target sequence is first bound to the detection probe and then bound to the capture oligonucleotide.

  United States Application No. 20050042608 (incorporated herein in its entirety) modifies “Method of Electrochemical Detection of Nucleic Acid Hybridization” (US Pat. No. 5,871,918) by Thorp et al. The method has been described. The presence or absence of a single nucleotide polymorphism located at an SNP site contained in a nucleic acid target can be examined by the method of United States Application No. 20050042608. Briefly, a plurality of capture probes are designed, and each capture probe includes a different SNP base and includes the probe base sequence on each side of the SNP base. The base of the probe is complementary to the corresponding target sequence adjacent to the SNP site. Each capture probe is secured to a separate electrode on the conductive working surface of the substrate and having a non-conductive outer layer. The degree of hybridization between each capture probe and the nucleic acid target is examined by detecting an oxidation-reduction reaction at each electrode using a transition metal. The difference in the oxidation rate at each electrode is used to determine whether the selected nucleic acid target has a single nucleotide polymorphism at the selected SNP site.

  Simultaneously determine the genotype of a large number of SNPs from a small or large pool of genomic material by the method of Lynx Therapeutics, Inc. (Hayward, Calif.) Using Megatype® technology Can do. This method uses a fluorescently labeled probe to compare two recovered genomic populations. As a result, it is possible to detect and recover DNA fragments containing SNP groups that distinguish between two populations without requiring prior knowledge of SNP mapping or SNP.

  There are many other ways to detect and identify SNPs. Among them, for example, there is a method of measuring a probe that hybridizes with SNP using mass spectrometry. This method varies in how quickly mass analysis can be performed, and can range from a few samples per day to a high throughput of 40,000 SNPs per day using mass code tags. One preferred embodiment is, for example, that a nucleic acid sequence containing the polymorphism of the present invention in the promoter of the COX2 gene, or a sequence complementary thereto, is examined by mass spectrometry. Such mass spectrometry is well known to those skilled in the art, and the genotyping method of the present invention can be modified to examine the polymorphism of the present invention (for example, the polymorphism of the present invention in the COX2 promoter) by mass spectrometry. Can be.

  SNPs can also be examined by ligation-bit analysis. This analysis requires two primers with a single base gap between the primers to be hybridized to the target. Each of the four nucleotides is added to a separate reaction mixture containing DNA polymerase, ligase, target DNA, and primers. The polymerase adds the 3 ′ end of the first primer complementary to the SNP to the nucleotide. The ligase then ligates two adjacent primers. When the sample is heated, if ligation has occurred, the now larger primer remains hybridized and, for example, fluorescence can be detected as a signal. Further details regarding these methods can be found in US Pat. Nos. 5,919,626, 5,945,283, 5,242,794, and 5,952,174.

  US Pat. No. 6,821,733 (incorporated herein in its entirety) is a method for detecting the difference in sequence of two nucleic acid molecules, wherein the two nucleic acid molecules are Contacting under conditions that allow complex formation and branch point migration; binding the quadruple complex with a tracer molecule and a detection molecule, and the detection molecule binding the tracer molecule or the quadruple complex. A method is described that includes contacting under possible conditions; and examining binding of the tracer molecule to the detection molecule before and after exposure to the quadruple complex. The competition between the quadruplex complex and the tracer molecule for binding to the detection molecule indicates that the two nucleic acids are different.

  Protein or proteomics based methods are also suitable for polymorphism detection and analysis. A polymorphism that causes a change in the expressed protein or that is associated with a change in the expressed protein can be detected directly by analyzing the protein. To do this, the various proteins in the sample are separated, for example by gel electrophoresis or HPLC, and the resulting protein or peptide is subjected to, for example, NMR or protein sequencing (for example, chemical sequencing or more common). Mass spectrometry). Proteomics-based methods are well known in the prior art and are likely to be automated. For example, integrated systems (eg Proteome Systems' Proteome IQ® system) combine sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technology. A platform for throughput proteome analysis is provided.

  The majority of proteomic methods for identifying proteins utilize mass spectrometry. For example, ion trap mass spectrometry, liquid chromatography (LC), LC / MSn mass spectrometry, gas chromatography (GC), Fourier transform-ion cyclo (R) tron resonance-mass spectrometer (FT-MS), MALDI -There are TOF mass spectrometry, ESI mass spectrometry, and variations of these. Mass spectrometry is also useful for investigating post-translational modifications of proteins (eg, phosphorylation, glycosylation), so polymorphisms that alter protein post-translational modifications and polymorphisms that are associated with changes in protein post-translational modifications To find out.

  Related techniques are also well known, such as protein processing devices such as “chemical ink jet printers”. This inkjet printer uses piezoelectric printing technology, and digesting a protein sample electroblotted from a 2-D PAGE gel onto a membrane in situ using enzymes or chemicals can be used to spot selected proteins. This is made possible by directly injecting an enzyme or chemical into the tube. After in situ digestion and incubation of the protein, the membrane can be placed directly into a mass analyzer for peptide analysis.

  One skilled in the art would know how to associate a particular SNP with altered gene expression, particularly when it occurs in a gene regulatory region such as a promoter. Changes in gene expression can also occur when the SNP is located in the coding region of a gene encoding a protein. For example, if SNPs are associated with various usage codons in the region, the SNPs are associated with differences in the amount of tRNA. Such changes in expression can be revealed by methods well known in the prior art and can be used to detect such SNPs. Similarly, if the SNP does not result in a similar amino acid substitution when it occurs in the coding region of a gene, the function of the gene product may change as a result of the substitution. Similarly, if the gene product is RNA, such SNPs can change the function of the RNA gene product. Any change in function can be examined, for example, in an activity assay or a functional assay, and the change can be used to detect such SNPs.

  The above methods for detecting and identifying SNPs can be used in the methods of the present invention.

  Of course, in order to detect and identify SNPs according to the present invention, a sample containing the material to be tested is taken from the subject. The sample can be any sample that may contain the desired SNP (and in some cases the desired polypeptide), including any body fluid (blood, urine, saliva, etc.), biopsy sample Or from other tissue preparations.

  DNA or RNA can be isolated from the sample by any of a number of methods known in the art. For example, nucleic acid purification methods are described in Tijssen, “Experimental Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part 1: Theory and Nucleic Acid Preparation”, Elsvia, New York, NY, 1993, Maniatis. , T., Fritsch, EF, Sambrook, J., “Molecular Cloning Manual”, 1989.

  Nucleic acid probes and / or nucleic acid primers may be provided to help detect the presence or absence of polymorphism / SNP. Such a probe is a substance that has a nucleic acid sequence specific for a chromosomal change that indicates the presence or absence of a polymorphism and emits a detectable signal when combined with the target polymorphism. It is preferable to label.

  The nucleic acid probe can be genomic DNA, cDNA, mRNA, any RNA-like material, and any DNA-like material (for example, peptide nucleic acid, branched DNA, etc.). The probe may be a sense polynucleotide probe or an antisense polynucleotide probe. If the target polynucleotide is double stranded, the probe can be a sense strand or an antisense strand. If the target polynucleotide is single stranded, the probe is a complementary single stranded.

  Probes can be prepared by various synthetic or enzymatic methods well known in the art. The probes can be synthesized in whole or in part by chemical methods well known in the prior art (Caruthers et al., Nucleic Acids Res., Symp. Ser., 215-233, 1980). Alternatively, the probe can be made in whole or in part using an enzyme.

  Nucleotide analogs can be incorporated into probes using methods well known in the art. The only condition is that the incorporated nucleotide analog must base pair with the target polynucleotide sequence. For example, some guanine nucleotides can be replaced with hypoxanthine, which forms base pairs with cytosine residues. However, these base pairs are not as stable as the base pairs between guanine and cytosine. Alternatively, adenine nucleotides can be replaced with 2,6-diaminopurine that forms a stronger base pair than that formed between adenine and thymidine.

  In addition, the probe can include chemically derivatized nucleotides or enzymatically derivatized nucleotides. Typical chemical modifications include derivatization using acyl groups, alkyl groups, aryl groups, and amino groups.

  The probe can be immobilized on a substrate. Preferred substrates are any of rigid or semi-rigid supports, specifically membranes, filters, chips, slides, wafers, fibers, magnetic beads, non-magnetic beads, gels, tubes , Plates, polymers, microparticles, capillaries, etc. The surface of the substrate can have various shapes (eg, wells, grooves, pins, channels, holes) to which polynucleotide probes are attached. The substrate preferably transmits light.

  Furthermore, the probe need not be directly attached to the substrate, but can be attached to the substrate through a linker group. The linker group is typically 6-50 atoms in length to expose the attached probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. The reactive group on the surface of the substrate reacts with one of the end portions of the linker to bond the linker to the substrate. The other end of the linker is then functionalized so that it can bind to the probe.

  The probe can be attached to the substrate by supplying a reagent for synthesizing the probe on the substrate surface, or by supplying a pre-formed DNA fragment or clone to the substrate surface. A typical dispenser is a micropipette that supplies a solution to a substrate. The position of the micropipette relative to the substrate is controlled using a robot system. A number of dispensers can be installed to allow the reagent to be supplied to multiple reaction zones simultaneously.

  Nucleic acid microarrays are preferred. Such microarrays (including nucleic acid chips therein) are well known in the prior art (eg, US Pat. Nos. 5,578,832, 5,861,242, 6,183,698, 6,287,850, 6,291,183, (See 6,297,018, 6,306,643, and 6,308,170, each of which is incorporated herein by reference).

  Alternatively, antibody microarrays can be made. Essential to how to make such a microarray is Schweitzer and Kingsmore, “Protein Measurement on a Microarray”, Curr. Opin. Biotechnol., 2002, 13 (1), 14-19; Avseekno Et al., "Immobilization of Proteins on Immunochemical Microarrays Produced by Electrospray Deposition", Anal. Chem., 2001, 15, 73 (24), 6047-6052; Huang, "Antibody-based protein Detection of multiple proteins in a microarray system, "Immunol. Methods, 2001, Vol. 255 (1-2), pages 1-13.

  The present invention also contemplates the manufacture of kits for use with the present invention. Suitable kits include various reagents used in the present invention in suitable containers or packaging materials (eg, tubes, vials, shrink wrap packages, blow molded packages).

  Suitable materials for inclusion in the kits of the present invention include one or more of the following: domains adjacent to the genetic polymorphism of interest in the DNA sequence or cDNA sequence domains; A pair of gene-specific PCR primers to be annealed (oligonucleotides); reagents that can amplify specific sequence domains in genomic DNA or cDNA without performing PCR; amplified by methods other than PCR or PCR Reagents necessary to distinguish between various alleles that may be present in different sequence domains (such as restriction endonucleases and oligonucleotides that preferentially anneal to one allele of a polymorphism (such as from oligonucleotides) Modified to include an enzyme or fluorescent chemical group that amplifies the signal, ensuring more allele discrimination)); Reagents required to physically separate products from various alleles (eg agarose or polyacrylamide and buffers used in electrophoresis, HPLC columns, SSCP gels, formamide gels, MALDI-TOF Matrix support).

  With the prediction method of the present invention, it is possible to evaluate whether a large number of treatments and / or treatment plans are suitable for a given object, and to achieve what is suitable for that object. The easiest way to do that would be to motivate the subject to change their lifestyle. For example, if the subject is currently a smoker, the method of the present invention can provide motivation to quit smoking.

  Therapeutic intervention or method of treatment is predicted from the nature of the polymorphism and the biological effects of that polymorphism. For example, if a susceptibility polymorphism is associated with a change in gene expression, intervention or therapy may involve restoring normal expression of the gene, for example by administration of a reagent capable of changing the expression of the gene. Preferably it is directed. If an allele or genotype of an SNP is associated with a decrease in gene expression, the treatment can include administering a reagent capable of increasing the expression of that gene, and conversely If the allele or genotype is associated with increased gene expression, the treatment can include administering a reagent capable of decreasing the expression of that gene. Methods that are useful for altering gene expression are well known in the prior art. For example, if an SNP allele or genotype is associated with upregulation of gene expression, for example, RNAi or antisense therapy can be used to reduce the amount of mRNA and consequently the gene Expression can be reduced. Alternatively, treatment can include a method of reversing the abnormal expression of the gene, eg, by altering the activity of the gene product.

  If a susceptible SNP allele or susceptible SNP genotype is associated with decreased function of the gene product, or decreased expression level of the gene product, therapeutic intervention or treatment includes operations that increase or replace its function Alternatively, it may include an operation of increasing the amount of the gene product in the body of the subject by, for example, administering the gene product or an equivalent function thereof. For example, where an SNP allele or genotype is associated with a decrease in enzyme function, the treatment can include administering an active enzyme or enzyme analog to the subject. Similarly, where an SNP allele or genotype is associated with increased function of a gene product, therapeutic intervention or treatment may, for example, reduce the level of the gene product, or an inhibitor of the gene product. The method may include an operation of administering a drug capable of reducing the function to a subject to reduce its function. For example, where an SNP allele or genotype is associated with increased enzyme function, the treatment can include administering an enzyme inhibitor to the subject.

  Similarly, if a beneficial (defensive) SNP is associated with upregulation of a particular gene or upregulation of an enzyme or other protein, treatment lacks a resistant genotype It is directed to mimic such upregulation or expression in an individual's body and / or to supply such an enzyme or other protein to the individual. In addition, if a protective SNP is associated with downregulation of a particular gene, or a decrease or disappearance of the expression of an enzyme or other protein, the preferred treatment is such in the body of an individual lacking the protective genotype. It is aimed at imitating various conditions.

  The relationship between the various polymorphisms identified above and the subject's susceptibility (or otherwise) to lung cancer also applies to the design and / or screening of candidate therapeutics. This is especially true when the relationship with a susceptibility or defensive polymorphism appears as an upregulation or downregulation of gene expression. In such cases, the effect of the candidate therapeutic on its upregulation or downregulation can be readily detected.

  For example, in one embodiment, existing human lung and cell cultures are screened for SNP genotypes as described above. (For information on human lungs and cell cultures, see, for example, Bohinski et al., 1996, Molecular and Cellular Biology, Vol. 14, pages 5671-5681; Collettsolberg et al., 1996, Pediatric Research, vol. 39, page 504; Hermanns 2004, Laboratory Investigation, Vol. 84, pages 736-752; Hume et al., 1996, In Vitro Cellular & Developmental Biology-Animal, Vol. 32, pages 24-29; Leonardi et al., 1995, Vol. 38 352-355; see Notingher et al., 2003, Biopolymers (Biospectroscopy), 72, 230-240; Ohga et al., 1996, Biochemical and Biophysical Research Communications, 228, 391-396. Note that each document is incorporated herein by reference in its entirety.) Select a culture that represents a group of susceptible and protective genotypes, and a protective genotype exists. If up or down A culture that is considered “normal” with respect to expression of the down-regulated gene is also selected.

  Samples of such cultures are checked against a library of candidate therapeutic compounds and screened for any or all of the following: (a) down-regulation of susceptibility genes that are normally up-regulated in susceptibility genotypes; (B) Up-regulation of susceptibility genes that are normally down-regulated in susceptibility genotypes; (c) Down-regulation of defense genes that are normally down-regulated or not expressed in defense genotypes (ie, do not express any form); (D) Upregulation of defense genes that are normally upregulated in the defense genotype. Compounds are selected for their ability to alter the regulatory state and / or action of susceptibility and / or defense genes in cultures with susceptibility genotypes.

  Similarly, a polymorphism that, when present, causes a physiologically active concentration of the expressed gene product to be outside the normal range for the subject, and the expressed gene product Individuals can be screened to see if they can benefit from their recovery measures if they can take preventive or therapeutic measures to return their levels to normal. Such screening includes the operation of detecting, by any method described herein, whether the polymorphism is present or absent in the subject. And it is judged that the subject in which the polymorphism exists may receive the benefit by treatment.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1]
Recruiting Case-Associated Research Subjects We have recruited subjects from Europe who have smoked at least 15 boxes per year and have been diagnosed by a physician with chronic obstructive pulmonary disease (COPD). Subject met the following criteria: Age> 50, symptoms of shortness of breath appearing after age 40, 1 second effort call volume (FEV1) less than 70% of expected, ratio FEV1 / FVC (forced call per second / forced vital capacity) is less than 79% (American Thoracic Society criteria). Recruited 294 subjects. Of these, 58% were male, the mean value of FEV1 / FVC (± 95% confidence interval) was 51% (49-53), and the mean value of FEV1 was 43% (41-45) of the expected value. Average age, number of cigarettes smoked per day, and box x year career were 65 years (64-66), 24 cigarettes / day (22-25), and 50 boxes x year (41-55), respectively. . Have smoked at least 20 boxes x years, have never been short of breath, have never been diagnosed with obstructive pulmonary disease in the past, and have been diagnosed with asthma or chronic obstructive pulmonary disease, especially in childhood We also examined 217 European subjects who did not. This control group was recruited through middle-aged clubs. 63% were male, FEV1 / FVC mean (95% confidence interval) was 82% (81-83), and FEV1 mean was 96% (95-97) of the expected value. Average age, number of cigarettes smoked per day, and box x year career were 59 years (57-61), 24 cigarettes / day (22-26), and 42 boxes x year (39-45), respectively. . Using a PCR-based method (Sanford et al., 1999), all subjects were examined for α1-antitrypsin (S and Z alleles) genotypes and subjects that were ZZ alleles were excluded. The COZ cohort and the resistant smokers cohort had the same proportion of subjects with MZ genotype (5% in each cohort). Continued recruitment of 190 European blood donors (smoking status unknown) through local blood donation services. 63% were male and the average age was 50 years.

  This study shows that polymorphisms that are more frequent in COPD patients compared to controls may reflect a greater susceptibility to developing pulmonary dysfunction and COPD. Similarly, polymorphisms that are more frequent in resistant smokers compared to susceptible smokers (COPD patients and / or controls) may play a protective role.

Genotyping method Cyclooxygenase 2 (COX2) promoter -765G / C polymorphism and α1-antitrypsin genotyping Genomic DNA was extracted from whole blood samples (Maniatis, T., Fritsch, EF, Sambrook, J , “Molecular Cloning Manual” (1989). The -765 polymorphism of cyclooxygenase 2 was altered in a slightly modified manner from a previously published method (Papafili, A. et al., 2002, the entirety of which is incorporated herein by reference). Examined. 20 ng genomic DNA, 500 pmoles forward and reverse primers, 0.2 mM dNTP, 10 mM Tris-HCl (pH 8.4), 150 mM KCl, 1.0 mM MgCl ₂ and 1 unit polymerase PCR reaction was performed in a total volume of 25 μl including (Life Technologies). The cycling time was 33 cycles of incubation at 95 ° C. for 3 minutes, followed by 94 ° C. for 50 seconds, 66 ° C. for 60 seconds, and 72 ° C. for 60 seconds. Thereafter, the final extension reaction was performed at 72 ° C. for 10 minutes. 4 μl of PCR product was visualized by irradiating 3% agarose gel stained with ethidium bromide with UV light. A 3 μl aliquot of the amplified product was digested with 4 units of AciI (Roche Diagnostics, New Zealand) at 37 ° C. for 1 hour. The digested product was separated over 2.0 hours at 80 mV on a 2.5% agarose gel using TBE buffer. The resulting product was visualized based on a 123 bp ladder by irradiating with ultraviolet light after staining with ethidium bromide. Using the PCR-based method described above (Sandford et al., 1999), S1- and Z-alleles of α1-antitrypsin among all smokers with COPD and resistant smokers The genotype of was examined.

Elafin + 49C / T polymorphism Genomic DNA was extracted from whole blood samples (Maniatis, T., Fritsch, EF, Sambrook, J., Molecular Cloning Manual, 1989). The +49 polymorphism of elafin has been previously published (Kuijpers, ALA et al., Clinical Genetics, 1998, 54, 96-101, which is incorporated herein by reference in its entirety. The +49 polymorphism of elafin was examined in a slightly modified manner. 20 ng genomic DNA, 500 pmoles forward and reverse primers, 0.2 mM dNTP, 10 mM Tris-HCl (pH 8.4), 150 mM KCl, 1.0 mM MgCl ₂ and 1 unit tack -The PCR reaction was carried out in a total volume of 25 µl containing polymerase (Life Technologies). The cycling time was 33 cycles of incubation at 95 ° C. for 3 minutes followed by 94 ° C. for 50 seconds, 66 ° C. for 60 seconds and 72 ° C. for 60 seconds. Thereafter, the final extension reaction was performed at 72 ° C. for 10 minutes. 4 μl of PCR product was visualized by irradiating 3% agarose gel stained with ethidium bromide with UV light. A 3 μl aliquot of the amplified product was digested with 4 units of Fok1 (Roche Diagnostics, New Zealand) at 37 ° C. for 1 hour. The digested product was separated over 2.0 hours at 80 mV on a 2.5% agarose gel using TBE buffer. The resulting product was visualized based on a 123 bp ladder by irradiating with ultraviolet light after staining with ethidium bromide.

Genotyping of the -1607 1G2G polymorphism of the matrix metalloproteinase 1 gene Genomic DNA was extracted by standard methods using phenol and chloroform. The patient and control cohorts were organized into a 96-well PCR format with a strategic negative control. Details of assay primers, PCR conditions, and RFLP assay have been reported previously (Dunleavey, L. et al.). Genotyping was performed with slight modifications to the above protocol to optimize it in our own laboratory conditions. Place PCR reaction in MJ Research Thermocycler, 80 ng genomic DNA, 100 ng forward primer and reverse primer, 200 mM dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl And 1.5 mM MgCl ₂ and 1.0 unit of Tack polymerase (Qiagen) were amplified in a total volume of 25 μl. The sequence of forward primer and reverse primer is 5'-TCG TGA GAA TGT CTT CCC ATT-3 '(SEQ ID NO: 1) and 5'-TCT TGG ATT GAT TTG AGA TAA GTG AAA TC-3' (SEQ ID NO: 2). there were. Cycling conditions were 35 cycles of 94 ° C. for 60 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds, and finally extended for 3 minutes. An aliquot of the amplified product was digested for 4 hours at the temperature specified by 6 units of restriction enzyme XmnI (Roche Diagnostics, New Zealand). The digested product was separated on a 6% polyacrylamide gel. After staining with ethidium bromide, this product was visualized by irradiation with ultraviolet rays, and compared with a 1 kb plus ladder (Invitrogen) based on the migration state. Genotypes were recorded in a data spreadsheet and statistically analyzed.

Genotyping of other polymorphisms Genomic DNA was extracted from whole blood samples (Maniatis, T., Fritsch, EF, Sambrook, J., Manual for Molecular Cloning, 1989). Place an aliquot of purified genomic DNA (concentration 10 ng / μl) in a 96-well plate and use the sequence, amplification conditions, and method shown below, and the Sequenom® system (Seakenom® Autoflex mass spectrometry) The genotype was determined on a vessel and a Samsung 24-pin nanodispenser).

In PCR multiplex reaction using the following conditions: The final concentration per 10 × buffer, 15 mM of MgCl ₂ and 1.25 ×, 25 mM of MgCl ₂ to 1.625MM, 500 [mu] M of dNTP mixture 25 mM, the primer 4 [mu] M 100 nM, Tack polymerase (Qiagen, Hot Start) was 0.15 unit / reaction, and genomic DNA was 10 ng / μl. The cycling time was 45 cycles of 95 ° C. for 15 minutes, 5 ° C. for 15 seconds, 56 ° C. for 30 seconds, 72 ° C. for 30 seconds, and finally extended for 3 minutes. Treat with shrimp alkaline phosphatase (SAP) (2 μl to 5 μl in one PCR reaction), incubate for 30 minutes at 35 ° C., then extend reaction (add 2 μl to 7 μl after treatment with SAP) Carried out. In the extension reaction, 0.76 μl of water; 0.2 μl of hME10 × stop buffer; 1 μl of hME primer (10 μM); 0.04 μl of mass extended enzyme was used for each reaction.

多型の遺伝子型を決定するためのシーケノムの条件-1

Sequenom's condition-1 for determining polymorphism genotype-1

多型の遺伝子型を決定するためのシーケノムの条件-2

Sequenom's condition for determining the genotype of a polymorphism -2

多型の遺伝子型を決定するためのシーケノムの条件-3

Sequenom's condition-3 for determining the genotype of polymorphism

多型の遺伝子型を決定するためのシーケノムの条件-4

Sequenom's condition for determining the genotype of polymorphism-4

多型の遺伝子型を決定するためのシーケノムの条件-5

Sequenom's condition for determining the genotype of polymorphism-5

多型の遺伝子型を決定するためのシーケノムの条件-6

Sequenom's condition for determining polymorphism genotype-6

多型の遺伝子型を決定するためのシーケノムの条件-7

Sequenom's condition for determining the genotype of polymorphism-7

多型の遺伝子型を決定するためのシーケノムの条件-8

Sequenom's condition for determining the genotype of polymorphism-8

*染色体の数（2n）遺伝子型
1．遺伝子型。抵抗力のある喫煙者とCOPDの患者でのCC/CG対GG、確率の比（OR）=1.98、95％信頼区間1.3〜3.1、χ²（イェーツの修正済み）=8.82、p=0.003、
CC/CG=COPDに対する防御
2．対立遺伝子。抵抗力のある喫煙者とCOPDの患者でのC対G、確率の比（OR）=1.92、95％信頼区間1.3〜2.8、χ²（イェーツの修正済み）=11.56、p<0.001、
C=COPDに対する防御 result

* Number of chromosomes (2n) genotype
1． Genotype. CC / CG vs GG in resistant smokers and COPD patients, probability ratio (OR) = 1.98, 95% confidence interval 1.3-3.1, χ ² (Yates corrected) = 8.82, p = 0.003,
Defense against CC / CG = COPD
2． Allele. C vs G in resistant smokers and COPD patients, ratio of probability (OR) = 1.92, 95% confidence interval 1.3-2.8, χ ² (Yates modified) = 11.56, p <0.001,
Defense against C = COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と対照でのGG対AG/AA、確率の比（OR）=1.83、95％信頼区間1.2〜2.8、χ²（イェーツの修正済み）=8.1、p=0.004、
GG=COPDに対する感受性（他のSNPの存在による）
2．対立遺伝子。抵抗力のある喫煙者と対照でのGG対AG/AA、確率の比（OR）=1.98、95％信頼区間1.3〜3.1、χ²（イェーツの修正済み）=9.43、p=0.002、
GG=COPDに対する防御（他のSNPの存在による）

* Number of chromosomes (2n)
1． Genotype. GG vs AG / AA in COPD patients and controls, probability ratio (OR) = 1.83, 95% confidence interval 1.2-2.8, χ ² (Yates corrected) = 8.1, p = 0.004,
Sensitivity to GG = COPD (due to the presence of other SNPs)
2． Allele. GG vs AG / AA in resistant smokers and controls, probability ratio (OR) = 1.98, 95% confidence interval 1.3-3.1, χ ² (Yates modified) = 9.43, p = 0.002,
Protection against GG = COPD (due to the presence of other SNPs)

*染色体の数（2n）
1．遺伝子型。COPDの患者と対照でのAA対AC/CC、確率の比（OR）=1.50、95％信頼区間1.0〜2.3、χ²（イェーツの修正済み）=4.26、p=0.04、
AA=COPDに対する感受性
2．対立遺伝子。COPDの患者と対照でのA対C、確率の比（OR）=1.46、95％信頼区間1.1〜2.0、χ²（イェーツの修正済み）=5.76、p=0.02
3．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA対AC/CC、確率の比（OR）=1.35、95％信頼区間0.9〜2.0、χ²（イェーツの修正済み）=2.39、p=0.12（傾向）、
AA=COPDに対する感受性

* Number of chromosomes (2n)
1． Genotype. AA vs. AC / CC in patients with COPD and control, probability ratio (OR) = 1.50, 95% confidence interval 1.0-2.3, χ ² (Yates corrected) = 4.26, p = 0.04,
Sensitivity to AA = COPD
2． Allele. A to C in patients with COPD and controls, probability ratio (OR) = 1.46, 95% confidence interval 1.1-2.0, χ ² (Yates corrected) = 5.76, p = 0.02
3． Genotype. AA to AC / CC in patients with COPD and resistant smokers, probability ratio (OR) = 1.35, 95% confidence interval 0.9-2.0, χ ² (Yates corrected) = 2.39, p = 0.12 ( Trend),
Sensitivity to AA = COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と対照でのCC対CG/GG、確率の比（OR）=1.44、95％信頼区間1.0〜2.2、χ²（イェーツの修正済み）=3.4、p=0.06、
CC=COPDに対する感受性
2．対立遺伝子。COPDの患者と対照でのC対G、確率の比（OR）=1.36、95％信頼区間1.0〜1.9、χ²（イェーツの修正済み）=53.7、p=0.05、
C=COPDに対する感受性

* Number of chromosomes (2n)
1． Genotype. CC vs. CG / GG in patients with COPD and control, probability ratio (OR) = 1.44, 95% confidence interval 1.0-2.2, χ ² (Yates corrected) = 3.4, p = 0.06,
Sensitivity to CC = COPD
2． Allele. C vs G in COPD patients and controls, probability ratio (OR) = 1.36, 95% confidence interval 1.0-1.9, χ ² (Yates corrected) = 53.7, p = 0.05,
Sensitivity to C = COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者での5G5G対それ以外、確率の比（OR）=1.55、95％信頼区間0.9〜2.6、χ²（イェーツの修正済み）=3.12、p=0.08、
5G5G=COPDに対する感受性
2．遺伝子型。COPDの患者と対照での5G5G対それ以外、確率の比（OR）=1.48、95％信頼区間0.9〜2.5、χ²（イェーツの修正済み）=2.43、p=0.12、
5G5G=COPDに対する感受性
3．対立遺伝子。COPDの患者と抵抗力のある喫煙者での5G対4G、確率の比（OR）=1.33、95％信頼区間1.0〜1.8、χ²（イェーツの修正済み）=4.02、p=0.05、
5G=COPDに対する感受性

* Number of chromosomes (2n)
1． Genotype. Probability ratio (OR) = 1.55, 95% confidence interval 0.9-2.6, χ ² (Yates corrected) = 3.12, p = 0.08, 5G5G vs. other smokers with COPD and resistant smokers
5G5G = Sensitivity to COPD
2． Genotype. Ratio of probability (OR) = 1.48, 95% confidence interval 0.9-2.5, χ ² (Yates corrected) = 2.43, p = 0.12
5G5G = Sensitivity to COPD
3． Allele. 5G vs 4G in COPD patients and resistant smokers, probability ratio (OR) = 1.33, 95% confidence interval 1.0-1.8, χ ² (Yates corrected) = 4.02, p = 0.05,
Sensitivity to 5G = COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者と対照でのTT対 TG/GG、確率の比（OR）=2.2、95％信頼区間1.0〜4.7、χ²（イェーツの修正済み）=4.49、p=0.03、
TT遺伝子型=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. TT vs TG / GG in resistant smokers and controls, probability ratio (OR) = 2.2, 95% confidence interval 1.0-4.7, χ ² (Yates corrected) = 4.49, p = 0.03,
TT genotype = protection against COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者とCOPDの患者でのAA/AC対CC、確率の比（OR）=1.39、95％信頼区間0.9〜2.1、χ²（イェーツの修正済み）=2.59、p=0.10、
AA/AC遺伝子型=COPDに対する防御
2．対立遺伝子。抵抗力のある喫煙者とCOPDの患者でのA対C、確率の比（OR）=1.34、95％信頼区間1.0〜1.8、χ²（イェーツの修正済み）=3.94、p=0.05、
A対立遺伝子=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. AA / AC versus CC in resistant smokers and patients with COPD, probability ratio (OR) = 1.39, 95% confidence interval 0.9-2.1, χ ² (Yates corrected) = 2.59, p = 0.10,
AA / AC genotype = protection against COPD
2． Allele. A to C in resistant smokers and COPD patients, probability ratio (OR) = 1.34, 95% confidence interval 1.0-1.8, χ ² (Yates corrected) = 3.94, p = 0.05,
A allele = protection against COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者と対照でのTT/TG対GG、確率の比（OR）=1.53、95％信頼区間1.0〜2.5、χ²（イェーツの修正済み）=3.52、p=0.06、
TT/TG遺伝子型=COPDに対する防御
2．対立遺伝子。抵抗力のある喫煙者と対照でのT対G、確率の比（OR）=1.27、95％信頼区間1.0〜1.7、χ²（イェーツの修正済み）=2.69、p=0.1、
T対立遺伝子=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. TT / TG vs GG in resistant smokers and controls, probability ratio (OR) = 1.53, 95% confidence interval 1.0-2.5, χ ² (Yates modified) = 3.52, p = 0.06,
TT / TG genotype = protection against COPD
2． Allele. T vs G in resistant smokers and controls, probability ratio (OR) = 1.27, 95% confidence interval 1.0-1.7, χ ² (Yates modified) = 2.69, p = 0.1,
T allele = protection against COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者と対照でのAA対AG/GG、確率の比（OR）=1.45、95％信頼区間0.9〜2.2、χ²（イェーツの修正済み）=3.19、p=0.07、
AA遺伝子型=COPDに対する防御
2．対立遺伝子。抵抗力のある喫煙者と対照でのA対G、確率の比（OR）=1.34、95％信頼区間1.0〜1.8、χ²（イェーツの修正済み）=3.71、p=0.05、
A対立遺伝子=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. AA vs AG / GG in resistant smokers and controls, probability ratio (OR) = 1.45, 95% confidence interval 0.9-2.2, χ ² (Yates modified) = 3.19, p = 0.07,
AA genotype = protection against COPD
2． Allele. A to G in resistant smokers and controls, probability ratio (OR) = 1.34, 95% confidence interval 1.0-1.8, χ ² (Yates modified) = 3.71, p = 0.05,
A allele = protection against COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と対照でのAA対AT/TT、確率の比（OR）=1.51、95％信頼区間0.9〜2.5、χ²（イェーツの修正済み）=3.07、p=0.08、
AA遺伝子型=COPDに対する感受性

* Number of chromosomes (2n)
1． Genotype. AA vs AT / TT in patients with COPD and control, probability ratio (OR) = 1.51, 95% confidence interval 0.9-2.5, χ ² (Yates corrected) = 3.07, p = 0.08,
AA genotype = susceptibility to COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者と対照でのAA対AG/GG、確率の比（OR）=2.94、95％信頼区間0.7〜14.0、χ²（イェーツの修正済み）=2.78、p=0.09、
AA遺伝子型=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. AA vs. AG / GG in resistant smokers and controls, probability ratio (OR) = 2.94, 95% confidence interval 0.7-14.0, χ ² (Yates corrected) = 2.78, p = 0.09,
AA genotype = protection against COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのTT対TC/CC、確率の比（OR）=6.03、95％信頼区間1.1〜42、χ²（イェーツの修正済み）=4.9、p=0.03、
TT遺伝子型=COPDに対する感受性

* Number of chromosomes (2n)
1． Genotype. TT vs TC / CC in COPD patients and resistant smokers, probability ratio (OR) = 6.03, 95% confidence interval 1.1-42, χ ² (Yates corrected) = 4.9, p = 0.03,
TT genotype = susceptibility to COPD

*染色体の数（2n）
1．遺伝子型。抵抗力のある喫煙者とCOPDの患者でのMS/SS対MM、確率の比（OR）=2.42、95％信頼区間1.2〜5.1、χ²（イェーツの修正済み）=5.7、p=0.01、
S=COPDに対する防御

* Number of chromosomes (2n)
1． Genotype. MS / SS vs. MM in resistant smokers and COPD patients, probability ratio (OR) = 2.42, 95% confidence interval 1.2-5.1, χ ² (Yates corrected) = 5.7, p = 0.01,
Defense against S = COPD

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA/AG対GG、確率の比（OR）=1.57、95％信頼区間0.9〜2.7、χ²（イェーツの修正済み）=2.52、p=0.11、
AA/AG=感受性（GG=防御）
2．対立遺伝子。COPDの患者と抵抗力のある喫煙者でのA対G、確率の比（OR）=1.61、95％信頼区間1.0〜2.7、χ²（イェーツの修正済み）=3.38、p=0.07、
A=感受性

* Number of chromosomes (2n)
1． Genotype. AA / AG vs. GG in patients with COPD and resistant smokers, probability ratio (OR) = 1.57, 95% confidence interval 0.9-2.7, χ ² (Yates corrected) = 2.52, p = 0.11,
AA / AG = Sensitivity (GG = Defense)
2． Allele. A to G in COPD patients and resistant smokers, probability ratio (OR) = 1.61, 95% confidence interval 1.0-2.7, χ ² (Yates corrected) = 3.38, p = 0.07,
A = Sensitivity

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対AG/AA、確率の比（OR）=0.77、95％信頼区間0.5〜1.2、χ²（イェーツの修正済み）=1.62、p=0.20、
GG=防御（AA/AG=感受性）傾向
2．対立遺伝子。COPDの患者と抵抗力のある喫煙者でのA対G、確率の比（OR）=1.3、95％信頼区間0.9〜1.9、χ²（イェーツの修正済み）=1.7、p=0.20、
A=感受性傾向

* Number of chromosomes (2n)
1． Genotype. GG vs AG / AA in COPD patients and resistant smokers, probability ratio (OR) = 0.77, 95% confidence interval 0.5-1.2, χ ² (Yates corrected) = 1.62, p = 0.20,
GG = defense (AA / AG = sensitivity) tendency
2． Allele. A vs G in COPD patients and resistant smokers, probability ratio (OR) = 1.3, 95% confidence interval 0.9-1.9, χ ² (Yates corrected) = 1.7, p = 0.20,
A = sensitivity tendency

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA/AG対GG、確率の比（OR）=0.26、95％信頼区間0.04〜1.4、χ²（イェーツの修正済み）=3.19、p=0.07、
AA/AG=防御（GG=感受性）

* Number of chromosomes (2n)
1． Genotype. AA / AG vs. GG in patients with COPD and resistant smokers, probability ratio (OR) = 0.26, 95% confidence interval 0.04-1.4, χ ² (Yates corrected) = 3.19, p = 0.07,
AA / AG = Defense (GG = Sensitivity)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対AG/AA、確率の比（OR）=1.60、95％信頼区間0.9〜2.7、χ²（イェーツの修正済み）=3.37、p=0.07、
GG=感受性
2．対立遺伝子。COPDの患者と抵抗力のある喫煙者でのG対A、確率の比（OR）=1.3、95％信頼区間1.0〜1.7、χ²（イェーツの修正済み）=2.90、p=0.09

* Number of chromosomes (2n)
1． Genotype. GG vs AG / AA in COPD patients and resistant smokers, probability ratio (OR) = 1.60, 95% confidence interval 0.9-2.7, χ ² (Yates corrected) = 3.37, p = 0.07,
GG = sensitivity
2． Allele. G to A in COPD patients and resistant smokers, probability ratio (OR) = 1.3, 95% confidence interval 1.0-1.7, χ ² (Yates modified) = 2.90, p = 0.09

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのCC/CG対GG、確率の比（OR）=3.2、95％信頼区間0.6〜22、χ²（イェーツの修正済み）=2.41、p=0.11（裾野が1つ）、
GC/CC=感受性（傾向）

* Number of chromosomes (2n)
1． Genotype. CC / CG vs. GG in patients with COPD and resistant smokers, probability ratio (OR) = 3.2, 95% confidence interval 0.6-22, χ ² (Yates corrected) = 2.41, p = 0.11 ( One skirt)
GC / CC = sensitivity (trend)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対それ以外、確率の比（OR）=0.53、95％信頼区間0.4〜0.80、χ²（イェーツの修正済み）=8.55、p=0.003、
GG=防御

* Number of chromosomes (2n)
1． Genotype. GG vs. other than smokers with COPD and resistant smokers, ratio of probability (OR) = 0.53, 95% confidence interval 0.4-0.80, χ ² (Yates corrected) = 8.55, p = 0.003,
GG = Defense

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA対AG/GG、確率の比（OR）=0.73、95％信頼区間0.5〜1.1、χ²（イェーツの修正済み）=1.91、p=0.17、
AA=防御傾向

* Number of chromosomes (2n)
1． Genotype. AA to AG / GG in COPD patients and resistant smokers, probability ratio (OR) = 0.73, 95% confidence interval 0.5-1.1, χ ² (Yates corrected) = 1.91, p = 0.17,
AA = defense trend

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA対AG/GG、確率の比（OR）=0.50、95％信頼区間0.2〜1.0、χ²（イェーツの修正済み）=3.82、p=0.05、
AA遺伝子型=防御

* Number of chromosomes (2n)
1． Genotype. AA vs. AG / GG in patients with COPD and resistant smokers, probability ratio (OR) = 0.50, 95% confidence interval 0.2-1.0, χ ² (Yates corrected) = 3.82, p = 0.05,
AA genotype = defense

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対AA/AG、確率の比（OR）=1.3、95％信頼区間0.9〜2.0、χ²（イェーツの修正済み）=1.86、p=0.17、
GG遺伝子型=感受性傾向

* Number of chromosomes (2n)
1． Genotype. GG vs AA / AG in patients with COPD and resistant smokers, probability ratio (OR) = 1.3, 95% confidence interval 0.9-2.0, χ ² (Yates corrected) = 1.86, p = 0.17,
GG genotype = susceptibility tendency

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのTT対CT/CC、確率の比（OR）=1.5、95％信頼区間1.0〜2.2、χ²（イェーツの修正済み）=3.51、p=0.06、
TT遺伝子型=感受性

* Number of chromosomes (2n)
1． Genotype. TT vs CT / CC in patients with COPD and resistant smokers, probability ratio (OR) = 1.5, 95% confidence interval 1.0-2.2, χ ² (Yates corrected) = 3.51, p = 0.06,
TT genotype = sensitivity

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対AA/AG、確率の比（OR）=0.64、95％信頼区間0.3〜1.4、χ²（イェーツの修正済み）=1.43、p=0.236、
GG遺伝子型=防御（傾向）

* Number of chromosomes (2n)
1． Genotype. GG vs AA / AG in COPD patients and resistant smokers, probability ratio (OR) = 0.64, 95% confidence interval 0.3-1.4, χ ² (Yates corrected) = 1.43, p = 0.236,
GG genotype = defense (trend)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA/AG対GG、確率の比（OR）=0.60、95％信頼区間0.3〜1.1、χ²（イェーツの修正済み）=2.34、p=0.12、
AA/AG遺伝子型=防御（GG=感受性）傾向

* Number of chromosomes (2n)
1． Genotype. AA / AG vs. GG in patients with COPD and resistant smokers, probability ratio (OR) = 0.60, 95% confidence interval 0.3-1.1, χ ² (Yates corrected) = 2.34, p = 0.12,
AA / AG genotype = defense (GG = sensitivity)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのCC/CT対TT、確率の比（OR）=2.0、95％信頼区間1.3〜3.1、χ²（イェーツの修正済み）=9.52、p=0.002、
CC/CT遺伝子型=感受性（TT=防御）

* Number of chromosomes (2n)
1． Genotype. CC / CT versus TT in COPD patients and resistant smokers, probability ratio (OR) = 2.0, 95% confidence interval 1.3-3.1, χ ² (Yates corrected) = 9.52, p = 0.002,
CC / CT genotype = sensitivity (TT = defense)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのAA対AT/TT、確率の比（OR）=1.7、95％信頼区間1.0〜2.7、χ²（イェーツの修正済み）=4.51、p=0.03、
AA遺伝子型=感受性

* Number of chromosomes (2n)
1． Genotype. AA vs AT / TT in patients with COPD and resistant smokers, probability ratio (OR) = 1.7, 95% confidence interval 1.0-2.7, χ ² (Yates corrected) = 4.51, p = 0.03,
AA genotype = susceptibility

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのCC対CT/TT、確率の比（OR）=1.4、95％信頼区間0.9〜2.2、χ²（イェーツの修正済み）=2.12、p=0.15、
CC遺伝子型=感受性（傾向）

* Number of chromosomes (2n)
1． Genotype. CC vs CT / TT in patients with COPD and resistant smokers, probability ratio (OR) = 1.4, 95% confidence interval 0.9-2.2, χ ² (Yates corrected) = 2.12, p = 0.15,
CC genotype = sensitivity (trend)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのCT/TT対CC、確率の比（OR）=0.70、95％信頼区間0.4〜1.3、χ²（イェーツの修正済み）=1.36、p=0.24、
CT/TT遺伝子型=防御（傾向のみ）
2．対立遺伝子。COPDの患者と抵抗力のある喫煙者でのT対C、確率の比（OR）=0.69、95％信頼区間0.4〜1.2、χ²（イェーツの修正済み）=1.91、p=0.17、
T遺伝子型=防御（傾向のみ）

* Number of chromosomes (2n)
1． Genotype. CT / TT vs. CC in patients with COPD and resistant smokers, probability ratio (OR) = 0.70, 95% confidence interval 0.4-1.3, χ ² (Yates corrected) = 1.36, p = 0.24,
CT / TT genotype = defense (trend only)
2． Allele. T vs C in COPD patients and resistant smokers, probability ratio (OR) = 0.69, 95% confidence interval 0.4-1.2, χ ² (Yates modified) = 1.91, p = 0.17,
T genotype = defense (trend only)

*染色体の数（2n）
1．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対CG/CC、確率の比（OR）=0.74、95％信頼区間0.4〜1.2、χ²（イェーツの修正済み）=1.47、p=0.23、
GG遺伝子型=防御（傾向）
2．遺伝子型。COPDの患者と抵抗力のある喫煙者でのGG対CG/CC、確率の比（OR）=0.69、95％信頼区間0.4〜1.2、χ²（イェーツの修正済み）=2.16、p=0.14、
GG遺伝子型=防御（傾向）

* Number of chromosomes (2n)
1． Genotype. GG vs CG / CC in COPD patients and resistant smokers, probability ratio (OR) = 0.74, 95% confidence interval 0.4-1.2, χ ² (Yates corrected) = 1.47, p = 0.23,
GG genotype = defense (trend)
2． Genotype. GG vs CG / CC in COPD patients and resistant smokers, probability ratio (OR) = 0.69, 95% confidence interval 0.4-1.2, χ ² (Yates corrected) = 2.16, p = 0.14,
GG genotype = defense (trend)

*染色体の数（2n）
1．遺伝子型。COPDの患者と対照での1G1G対それ以外、確率の比（OR）=0.43、95％信頼区間0.3〜0.7、χ²（イェーツの修正済み）=13.3、p=0.0003、
1G1G遺伝子型=防御
2．対立遺伝子。COPDの患者と対照での1G対2G、確率の比（OR）=0.45、95％信頼区間0.3〜0.6、χ²（イェーツの修正済み）=28.8、p<0.0001、
1G=防御
3．遺伝子型。COPDの患者と抵抗力のある喫煙者での1G1G/1G2G対それ以外、確率の比（OR）=0.55、95％信頼区間0.4〜0.9、χ²（イェーツの修正済み）=6.83、p=0.009、
1G1G/1G2G遺伝子型=防御
4．対立遺伝子。COPDの患者と抵抗力のある喫煙者での1G対2G、確率の比（OR）=0.73、95％信頼区間0.6〜1.0、χ²（イェーツの修正済み）=4.61、p=0.03、
1G=防御
5．遺伝子型。COPDの患者と対照での2G2G対1G1G/1G2G、確率の比（OR）=3.17、95％信頼区間1.9〜5.3、χ²（イェーツの修正済み）=21.4、p<0.0001、
2G2G遺伝子型=感受性
6．対立遺伝子。COPDの患者と対照での2G対1G、確率の比（OR）=2.2、95％信頼区間1.6〜3.0、χ²（イェーツの修正済み）=28.8、p<0.00001、
2G=感受性
7．遺伝子型。COPDの患者と抵抗力のある喫煙者での2G2G対1G1G/1G2G、確率の比（OR）=1.81、95％信頼区間1.2〜2.9、χ²（イェーツの修正済み）=6.83、p=0.009、
2G2G遺伝子型=感受性
8．対立遺伝子。COPDの患者と抵抗力のある喫煙者での2G対1G、確率の比（OR）=1.4、95％信頼区間1.0〜1.8、χ²（イェーツの修正済み）=4.61、p=0.0003、
2G=感受性

* Number of chromosomes (2n)
1． Genotype. 1G1G vs. other than COPD patients and controls, probability ratio (OR) = 0.43, 95% confidence interval 0.3-0.7, χ ² (Yates corrected) = 13.3, p = 0.0003,
1G1G genotype = defense
2． Allele. 1G vs 2G in COPD patients and controls, probability ratio (OR) = 0.45, 95% confidence interval 0.3-0.6, χ ² (Yates corrected) = 28.8, p <0.0001,
1G = Defense
3． Genotype. Probability ratio (OR) = 0.55, 95% confidence interval 0.4-0.9, χ ² (Yates corrected) = 6.83, p = 0.009 for COPD patients and resistant smokers ,
1G1G / 1G2G genotype = defense
Four. Allele. 1G vs 2G in patients with COPD and resistant smokers, probability ratio (OR) = 0.73, 95% confidence interval 0.6-1.0, χ ² (Yates corrected) = 4.61, p = 0.03,
1G = Defense
Five. Genotype. 2G2G vs. 1G1G / 1G2G in COPD patients and controls, probability ratio (OR) = 3.17, 95% confidence interval 1.9 to 5.3, χ ² (Yates corrected) = 21.4, p <0.0001,
2G2G genotype = sensitivity
6． Allele. 2G vs 1G in COPD patients and controls, probability ratio (OR) = 2.2, 95% confidence interval 1.6-3.0, χ ² (Yates corrected) = 28.8, p <0.00001,
2G = Sensitivity
7． Genotype. 2G2G vs. 1G1G / 1G2G in COPD patients and resistant smokers, probability ratio (OR) = 1.81, 95% confidence interval 1.2-2.9, χ ² (Yates corrected) = 6.83, p = 0.009,
2G2G genotype = sensitivity
8． Allele. 2G to 1G in COPD patients and resistant smokers, probability ratio (OR) = 1.4, 95% confidence interval 1.0-1.8, χ ² (Yates corrected) = 4.61, p = 0.0003,
2G = Sensitivity

Discussion From the above results, it can be seen that several polymorphisms are associated with susceptibility and / or resistance to obstructive pulmonary disease in persons exposed to smoking environments. Individual polymorphisms alone do not appear to provide acceptable disease predictions, albeit with differences. These polymorphisms, when combined, distinguish sensitive smokers (being COPD) from resistant smokers. These polymorphisms include the promoter polymorphism (which alters gene expression and hence protein synthesis) and the exon polymorphism (amino acid sequence in a process known to cause lung remodeling). Change (known to change expression and / or function)). The polymorphisms identified in this specification are found in genes that encode proteins that play a central role in processes such as inflammation, matrix remodeling, oxidative stress.

  By comparing smokers with COPD and smokers with nearly normal lung function, some polymorphisms may be significantly more or less than the comparison group (including the blood donor cohort) It was revealed.

  Analysis of the -765 C / G polymorphism of the cyclooxygenase 2 gene promoter found that the C allele and CC / CG genotype were significantly higher in the resistant smoker cohort than in the COPD cohort (OR = 1.92, P <0.001, OR = 1.98, P = 0.003). This is consistent with the role of defense. More frequent than the blood donor cohort also indicates that the C allele (CC genotype) accounts for a large proportion of resistant groups (see Table 1).

  Analysis of the 16 arginine / glycine polymorphism of the β2 adrenergic receptor gene found that the GG genotype was significantly higher than the control in the COPD cohort (OR = 1.83, P = 0.004). This indicates that this genotype and susceptibility to smoking may be related. GG genotypes also make up a large proportion of resistant cohorts, but their effects may be weaker than those of protective polymorphisms (see Table 2).

  Analysis of the 105 C / A polymorphism of interleukin 18 gene found that the A allele and AA genotype were significantly higher than the control in the COPD cohort (OR = 1.46, P = 0.02, respectively) OR = 1.50, P = 0.04). This is consistent with the role of sensitivity. The AA genotype was also more than the cohort of resistant smokers in the COPD cohort (OR = 1.35, P = 0.12). This trend is consistent with the role of sensitivity (see Table 3a).

  Analysis of the -133 G / C polymorphism of the IL18 promoter found that the C allele and CC genotype were significantly higher than the control in the COPD cohort (OR = 1.36, P = respectively) 0.05, OR = 1.44, P = 0.06). This is consistent with the role of sensitivity. The CC genotype was also more than the COPD cohort of resistant smokers. This trend is consistent with the role of sensitivity (see Table 3b).

  Analysis of the -675 4G / 5G promoter of the plasminogen activator inhibitor gene found that the 5G allele and 5G5G genotype were significantly higher than the cohort of resistant smokers in the COPD cohort (OR = 1.33, P = 0.05, OR = 1.55, P = 0.08, respectively). This is consistent with the role of sensitivity. The higher frequency of 5G5G in the COPD cohort than in the blood donor cohort also indicates that the 5G5G genotype is associated with susceptibility (see Table 4).

  When analyzing the 298 aspartate / glutamate (T / G) polymorphism of the NO synthase 3 (NOS3) gene, the TT genotype is a cohort of resistant smokers and a cohort of blood donors (OR = 2.2, P = 0.03) was found to be significantly higher. This is consistent with the role of defense (see Table 5).

  Analysis of the ricin 420 threonine (A / C) polymorphism of the vitamin D binding protein gene reveals that there are more A alleles and AA / AC genotypes in the resistant smoker cohort than in the COPD cohort (OR = 1.34, P = 0.05, OR = 1.39, P = 0.10, respectively). This is consistent with the role of defense (see Table 6a).

  Analysis of the glutamic acid 416 aspartic acid (T / G) polymorphism of the vitamin D binding protein gene has more T alleles and TT / TG genotypes in the resistant smoker cohort than the blood donor cohort (OR = 1.27, P = 0.10, OR = 1.53, P = 0.06, respectively). This is consistent with the role of defense (see Table 6b).

  Analysis of the isoleucine 105 valine (A / G) polymorphism of the glutathione S transferase P gene reveals that there are more A alleles and AA genotypes in the resistant smoker cohort than in the blood donor cohort (OR = 1.34, P = 0.05, OR = 1.45, P = 0.07, respectively). This is consistent with the role of defense (see Table 7).

  Analysis of the 874 A / T polymorphism of the IFN-γ gene found that the AA genotype was significantly higher than the control in the COPD cohort (OR = 1.51, P = 0.08). This is consistent with the role of sensitivity (see Table 8).

  Analysis of the 130 arginine / glutamine (G / A) polymorphism of the interleukin 13 gene found that the AA genotype was higher in the resistant smoker cohort than in the blood donor cohort (OR = 2.94, P = 0.09). This is consistent with the role of defense (see Table 9a).

  Analysis of the -1055 C / T polymorphism of the interleukin 13 gene found that the TT genotype was higher than the cohort of resistant smokers in the COPD cohort (OR = 6.03, P = 0.03) ). This is consistent with the role of sensitivity (see Table 9b).

  Analysis of the α1-antitrypsin S polymorphism found that there were more S alleles and MS / SS genotypes in the resistant smokers cohort than in the COPD cohort (OR = 2.42, P = 0.01). This is consistent with the role of defense (see Table 10).

  When analyzing the +489 G / A polymorphism of the tumor necrosis factor α gene, the A allele and the AA / AG genotype are cohorts of resistant smokers in the COPD cohort (OR = 1.57, P = 0.11 ) Was found. This is consistent with the role of sensitivity (see Table 11a). Conversely, GG genotypes were found to be more common in resistant smokers cohorts. This is consistent with the role of defense (see Table 11a).

  Analysis of the -308 G / A polymorphism within the tumor necrosis factor alpha gene promoter found that the GG genotype was higher in the cohort of resistant smokers than in the COPD cohort (OR = 0.77) , P = 0.20). This is consistent with the role of defense (see Table 11b). Conversely, the A allele, AA and AG genotypes were found to be more common in the COPD cohort (OR = 1.3, P = 0.20). This is consistent with the role of sensitivity (see Table 11b).

  ・ Analysis of the C89Y A / G polymorphism of the SMAD3 gene found that the AA / AG genotype was higher in the cohort of resistant smokers than the COPD cohort (OR = 0.26, P = 0.07) . This is consistent with the role of defense (see Table 12). Conversely, GG genotypes were found to be more common in the COPD cohort. This is consistent with the role of sensitivity (see Table 12).

  Analysis of the E469K A / G polymorphism of the intercellular adhesion molecule 1 gene found that there were more G alleles and GG genotypes than the cohort of resistant smokers in the COPD cohort (respectively, OR = 1.3, P = 0.09, OR = 1.60, P = 0.07). This is consistent with the role of sensitivity (see Table 13).

  Analysis of the glycine 881 arginine G / C polymorphism of the caspase (NOD2) gene found that the CC / CG genotype was higher than the cohort of resistant smokers in the COPD cohort (OR = 3.2) , P = 0.11). This is consistent with the role of sensitivity (see Table 14).

  Analysis of the 161 G / A polymorphism of the mannose-binding lectin 2 gene found that the GG genotype was higher in the resistant smokers cohort than the COPD cohort (OR = 0.53, P = 0.003) ). This is consistent with the role of defense (see Table 15).

  Analysis of the -1903 G / A polymorphism of the chymase 1 gene found that the AA genotype was higher in the resistant smokers cohort than in the COPD cohort (OR = 0.73, P = 0.17) . This is consistent with the role of defense (see Table 16).

  Analysis of the arginine 197 glutamine G / A polymorphism of the N-acetyltransferase 2 gene found that the AA genotype was higher in the cohort of resistant smokers than in the COPD cohort (OR = 0.50, P = 0.05). This is consistent with the role of defense (see Table 17).

  Analysis of the -511 A / G polymorphism of the interleukin 1B gene found that the GG genotype was greater than the cohort of resistant smokers in the COPD cohort (OR = 1.3, P = 0.17) ). This is consistent with the role of sensitivity (see Table 18).

  Analysis of the tyrosine 113 histidine T / C polymorphism of the microsomal epoxide hydrolase gene found that the TT genotype was higher than the cohort of resistant smokers in the COPD cohort (OR = 1.5, P = 0.06). This is consistent with the role of sensitivity (see Table 19a).

  Analysis of the arginine 139 G / A polymorphism of the microsomal epoxide hydrolase gene found that the GG genotype was higher in the cohort of resistant smokers than in the COPD cohort (OR = 0.64, P = 0.23). This is consistent with the role of defense (see Table 19b).

  Analysis of the -366 G / A polymorphism of the 5 lipo-oxygenase gene found that the AG / AA genotype was higher in the cohort of resistant smokers than the COPD cohort (OR = 0.60, P = 0.12). This is consistent with the role of defense (see Table 20). Conversely, GG genotypes were found to be more common in the COPD cohort. This is consistent with the role of sensitivity (see Table 20).

  ・ Analysis of the HOM T2437C polymorphism of the heat shock protein 70 gene found that the CC / CT genotype was higher than the cohort of resistant smokers in the COPD cohort (OR = 2.0, P = 0.002) It was. This is consistent with the role of sensitivity (see Table 21). Conversely, TT genotypes were found to be more common in resistant smokers cohorts. This is consistent with the role of defense (see Table 21).

  When analyzing the +13924 T / A polymorphism of the calcium-dependent chloride channel 1 gene, the AA genotype is higher than the cohort of resistant smokers in the COPD cohort (OR = 1.7, P = 0.03) I found something. This is consistent with the role of sensitivity (see Table 22).

  Analysis of the -159 C / T polymorphism of the monocyte differentiation antigen CD-14 gene showed that the CC genotype was higher in the COPD cohort than in the smoker cohort (OR = 1.4, P = 0.15) ). This is consistent with the role of sensitivity (see Table 23).

  Analysis of the exon 1 +49 C / T polymorphism of the elafin gene found that there were more T alleles and CT / TT genotypes in the resistant smoker cohort than the COPD cohort ( OR = 0.69, P = 0.17, OR = 0.70, P = 0.24, respectively. This is consistent with the role of defense (see Table 24).

  Analysis of the glutamine 27 glutamate C / G polymorphism of the β2 adrenergic receptor gene found that the GG genotype was higher in the resistant smoker and blood donor cohorts than in the COPD cohort (OR = 0.74, P = 0.23, OR = 0.69, P = 0.14, respectively). This is consistent with the role of defense (see Table 25).

  Analysis of the -1607 1G / 2G polymorphism of the MMP1 gene promoter found that the 1G allele and 1G1G / 1G2G genotype were significantly higher in the resistant smoker cohort than in the COPD cohort (OR = 0.73, P = 0.03, OR = 0.55, P = 0.009, respectively). This is consistent with the role of defense. The higher frequency of 1G1G in the blood donor cohort than in the COPD cohort also indicates that the 1G allele is protective (see Table 26).

  The trend for chronic obstructive pulmonary disease (eg emphysema and COPD) is a result of a combination of individual genetic composition and lifelong exposure to various air pollution, of which smoking is the most common It is accepted. Similarly, COPD is accepted to include several obstructive pulmonary diseases and to be characterized by impaired expiratory flow rate (eg, FEV1). The data presented in this specification suggests that several genes may be involved in the development of COPD. Numerous genetic mutations acting in combination to promote or protect against lung damage may be associated with increased resistance or sensitivity.

From the analysis of individual polymorphisms, 19 protective genotypes were identified and their frequency was analyzed in a cohort of smokers consisting of resistant smokers and smokers suffering from COPD. Resistant smokers and smokers suffering from COPD with protective genotypes (CC / CG of COX2, arginine 16 glycine AA of β2 adrenergic receptor, arginine 130 glutamine AA of interleukin 13, 298TT of NO synthase 3, A significant difference was found when comparing the frequency of 0, 1, and 2+ vitamin D binding proteins (selected from 420AA / AC) (total χ ² = 16.43, P = 0.0003) . This is because smokers with two or more protective genotypes are more than 3 times more resistant (OR = 3.1, P = 0.004), whereas smokers without protective genotypes It shows that it is likely to be about twice as high as COPD (OR = 1.74, P = 0.006) (see Table 28). When examined differently, the chance of becoming COPD decreased to 63%, 55%, 32%, etc. for smokers with 0, 1 and 2+ tested genotypes, respectively. . Another defense genotype selected (in COX2 CC / CG, NOS3 298TT, VDBP -420AA / AC, VDBP -416TT / TG, GSTP1 AA, IL13 -140AA, α1-AT MS / SS Analysis), there was a significant difference in the frequency of COPD smokers and resistant smokers between 0 and 1 tested genotypes (OR = 2.82). , P = 0.0004) (See Table 30). This indicates that zero defense genotypes tested are likely to be 2-3 times more likely to COPD.

Based on the analysis of individual polymorphisms, 17 susceptibility genotypes were identified and their frequency was analyzed in a cohort of smokers consisting of resistant smokers and smokers suffering from COPD. Resistant smokers and smokers suffering from COPD are susceptible genotypes (choose from interleukin 18 105AA, PAI-1 -675 5G5G, interleukin 13 -1055TT, interferon gamma -874TT) When the frequencies of 0, 1, and 2+ were compared, a significant difference was found (overall χ ² = 8.72, P = 0.01). This means that smokers with two or more tested susceptibility genotypes are more than twice as likely to have COPD (OR = 1.9, P = 0.009) versus one tested susceptibility genotype. Smokers are likely to be 1.5 times more likely to COPD (OR = 1.5, P = 0.05) (see Table 29). When examined differently, the chance of becoming COPD increased to 49%, 55%, 68%, etc. for smokers with 0, 1, or 2+ susceptibility genotypes examined. .

  These findings indicate that the method of the present invention can predict COPD and / or emphysema long before symptoms appear for an individual.

  These findings thus also provide opportunities for therapeutic intervention and / or treatment planning, as already explained. Briefly, such interventions or plans can include preparing a subject for a lifestyle change or a therapy that normalizes abnormal gene expression or gene product function. For example, the -765G allele within the promoter of the gene encoding COX2 is associated with more expression than is observed with the C allele. As indicated herein, the C allele is a protective factor for the predisposition or potential risk of developing COPD and / or emphysema. Therefore, a suitable treatment for a subject known to have the -765G allele can be the administration of a drug capable of reducing the expression of the gene encoding COX2. As another suitable treatment, a COX2 inhibitor can be administered to such subjects, for example, with additional therapy, gene therapy, RNAi, and the like. In another example, as demonstrated herein, the -133C allele within the promoter of the gene encoding IL18 is associated with susceptibility to COPD and / or emphysema. Because the -133C allele within the promoter of the gene encoding IL18 is associated with elevated levels of IL18, a suitable treatment for subjects known to have the -133C allele encodes IL18 It is possible to administer a drug capable of increasing the expression of the active gene. In yet another example, as indicated herein, the -675 5G5G genotype within the promoter of the plasminogen activator inhibitor gene is associated with susceptibility to COPD and / or emphysema. The 5G allele has been reported to be associated with increased repressor protein binding and decreased transcription of the gene. One suitable treatment may include administration of an agent capable of reducing the level of repressor and / or an agent capable of blocking repressor binding. This relieves the effect of the repressor down-regulating transcription. Another therapy is gene therapy, for example, introducing at least one copy of a plasminogen activator inhibitor gene with reduced binding affinity to a repressor (eg, a copy of a gene with the -675 4G4G genotype) It is possible.

  Suitable methods and agents for use in such treatment are well known in the prior art and are described in this specification.

  Once both sensitive and protective polymorphisms have been identified as described in this specification, candidate compounds are screened to assess the effects of the candidate compounds in prophylactic and / or therapeutic approaches Opportunities are also provided. Such screening methods can determine whether any of a wide variety of candidate compounds have the ability to reverse or compensate for genotypic or phenotypic effects of susceptibility polymorphisms or genotypic or phenotypic effects of protective polymorphism It includes operations that reveal whether you have the ability to imitate or reproduce.

  Further provided are methods for assessing a subject's responsiveness to available prevention or treatment methods. The method is applied when the applicable therapy is to restore the physiologically active concentration of the expressed gene product from a condition that is larger or smaller than the normal range for the subject's age and gender. Especially applied. In that case, the method includes an operation to detect the presence or absence of the susceptible polymorphism. When present, gene expression is up- or down-regulated, resulting in an over or deficient state. A subject with the polymorphism can then respond to treatment.

References

Maniatis, T., Fritsch, EF, Sambrook, J., “Molecular Cloning Manual”, 1989.
Kuijpers, ALA, Pfundt, R., Zeeuwen, LJM., Et al., “The genetic polymorphism of SKALP / elafin is not related to the pustular morphology of psoriasis”, Clin. Genetics, 1998, 54, 96- 101 pages.
Papafili, A. et al., 2002. “A common promoter mutation in cyclooxygenase-2 suppresses gene expression,” Arterioscler. Thromb. Vasc. Biol., Vol. 20, pp. 1631-1635.
Sandford, AJ et al., 1999, Z- and S-mutations of the α1-antitrypsin gene and the risk of chronic obstructive pulmonary disease, Am. J. Respir. Cell. Mol. Biol., Volume 20, 287-291 pages.
Waltenberg, J., 2001, “Pathophysiological basis of unstable coronary syndrome”, Herz, Vol. 26, Supplement 1, pages 2-8.

  All publications, journal articles, other references and materials cited or mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains, and each such reference or material is , Incorporated herein by reference in its entirety as if individually incorporated by reference or as if disclosed in its entirety in this specification. Applicants must physically include all such patents, publications, academic papers, websites, electronically available information, and any material and information from other reference materials and documents in this specification. Has the right to incorporate.

  The specific methods and compositions described herein are merely illustrative of various embodiments or preferred embodiments and are not intended to limit the scope of the invention. Other objects, features, examples, and embodiments will be within the spirit of the invention as defined by the scope of the claims, since those skilled in the art will recognize from this specification. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed in this specification without departing from the scope and spirit of the invention. Although the invention specifically described in this specification may be properly practiced in the absence of any elements or limitations, it is specifically disclosed in this specification that this is important. Absent. For example, in each case, embodiment, and example of the present invention, the expressions “comprising”, “consisting mainly of”, and “consisting of” can all be replaced by the other two in this specification. As such, it is illustrative of various alternative embodiments of the invention that have different ranges. In addition, expressions such as “comprising”, “including”, “containing” should be read without limitation. The methods and processes specifically described in this specification can be performed by changing the order of steps, and are not limited to the order of steps shown in this specification and claims. In this specification and the appended claims, the singular forms “a” and “an” include the plural unless the context clearly dictates otherwise. For example, reference to “a host cell” includes plural forms of such host cells (eg, cultures or populations). This patent should not be construed as limited to the examples, embodiments, and methods specifically disclosed in this specification. Interpreting this patent as limited by statements by the examiner or other officials of the Patent and Trademark Office is not subject to specific restrictions or reservations expressly adopted by the applicant in the response.

  The terms and expressions used are used for explanation and are not intended to limit the scope, so when such terms and expressions are used, they are equivalent to the stated expressions or parts thereof All expressions are not excluded and various modifications are possible within the scope of the invention as defined in the claims. Thus, although this specification specifically discloses the invention based on preferred embodiments and optional features, those skilled in the art will be able to conceive various modifications and changes to the concept disclosed herein. Let's go. Such modifications and changes are considered to be within the scope of the present invention as defined in the appended claims.

It is the graph which plotted the ratio of the person suffering from COPD with respect to the number of the mutated defense genes. It is the graph which plotted the ratio of the person suffering from COPD with respect to the number of the susceptibility genes which were mutated. There is a linear relationship here, and it is estimated that as high as 80% of people with four or more mutated susceptibility genes become COPD.

Claims (53)

A method to determine whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or a method of diagnosing the likelihood of developing COPD and / or emphysema And
-765 C / G in the promoter of the gene encoding COX2;
105 C / A within the gene encoding IL18;
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G within the promoter of the gene encoding PAI-1;
874 A / T in the gene encoding IFN-γ;
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14);
Exon 1 +49 C / T in the gene encoding elafin;
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding MMP1;
A polymorphism in linkage disequilibrium with any of the above polymorphisms;
Including analyzing one or more polymorphisms selected from the group consisting of:
A method wherein the genotype indicates a predisposition and / or potential risk of developing COPD and / or emphysema in the subject.   The above analysis is or includes an analysis of the -765 C / G polymorphism in the promoter of the gene encoding cyclooxygenase 2 and / or a polymorphism in linkage disequilibrium with this polymorphism 2. The method of claim 1, wherein   The above analysis is or includes an analysis of a 105 C / A polymorphism within the gene encoding interleukin 18 and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1.   The above analysis is or is an analysis of the -133 G / C polymorphism in the promoter of the gene encoding interleukin 18, and / or a polymorphism in linkage disequilibrium with this polymorphism. 2. The method of claim 1 comprising.   The above analysis is an analysis of the -675 4G / 5G polymorphism within the promoter of the gene encoding plasminogen activator inhibitor 1, and / or a polymorphism in linkage disequilibrium with this polymorph, or 2. The method of claim 1, comprising the analysis.   The analysis is or includes an analysis of a 874 A / T polymorphism in the gene encoding interferon γ and / or a polymorphism in linkage disequilibrium with the polymorphism, The method of claim 1.   The analysis is, or includes, an analysis of a 489 G / A polymorphism within the gene encoding tumor necrosis factor α and / or a polymorphism in linkage disequilibrium with this polymorphism. The method of claim 1 wherein:   The analysis is or includes an analysis of a C89Y A / G polymorphism within the gene encoding SMAD3 and / or a polymorphism in linkage disequilibrium with this polymorphism. Item 2. The method according to Item 1.   The above analysis is, or includes, an analysis of the E469K A / G polymorphism in the gene encoding intercellular adhesion molecule 1 and / or a polymorphism in linkage disequilibrium with this polymorphism. 2. The method of claim 1, wherein   The above analysis is or includes an analysis of the glycine 881 arginine polymorphism in the gene encoding caspase (NOD2) and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1.   The above analysis is, or includes, analysis of a 161 G / A polymorphism in the gene encoding mannose-binding lectin 2 and / or a polymorphism in linkage disequilibrium with this polymorphism. The method of claim 1 wherein:   The analysis is or includes an analysis of the -1903 G / A polymorphism within the gene encoding chymase 1 and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1.   The above analysis is or is an analysis of the arginine 197 glutamine G / A polymorphism in the gene encoding N-acetyltransferase 2 and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1, comprising:   The above analysis is, or includes, an analysis of the -366 G / A polymorphism in the gene encoding 5 lipo-oxygenase and / or a polymorphism in linkage disequilibrium with this polymorphism 2. The method of claim 1, wherein   The above analysis is or includes an analysis of a HOM T2437C polymorphism in the gene encoding heat shock protein 70 and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1.   The analysis is for the +13924 T / A polymorphism in the gene encoding calcium-dependent chloride channel 1 and / or a polymorphism in linkage disequilibrium with this polymorph, or 2. The method of claim 1, comprising analysis.   The analysis is for the -159 C / T polymorphism in the gene encoding the monocyte differentiation antigen CD-14 and / or a polymorphism in linkage disequilibrium with this polymorph, or 2. The method of claim 1, comprising analysis.   The above analysis is or includes an analysis of exon 1 +49 C / T polymorphism in the gene encoding elafin and / or a polymorphism in linkage disequilibrium with this polymorphism The method of claim 1 wherein:   The above analysis is an analysis of the -1607 1G / 2G polymorphism within the promoter of the gene encoding MMP1 (for the 1G allele only) and / or a polymorphism in linkage disequilibrium with this polymorphism 2. The method of claim 1 comprising an analysis thereof. The above analysis
16 arginine / glycine in the gene encoding the β2 adrenergic receptor;
130 arginine / glutamine (G / A) within the gene encoding interleukin 13 (IL13);
298 aspartate / glutamate (T / G) in the gene encoding NO synthase 3 (NOS3);
Isoleucine 105 valine (A / G) in the gene encoding glutathione S-transferase P (GST-P);
Glutamic acid 416 aspartic acid (T / G) in the gene encoding vitamin D binding protein (VDBP);
Lysine 420 threonine (A / C) in the gene encoding vitamin D binding protein;
-1055 C / T in the promoter of the gene encoding interleukin-13;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G in the promoter of the gene encoding interleukin 1B (IL1B);
Tyrosine 113 histidine T / C in the gene encoding microsomal epoxide hydrolase (MEH);
Arginine 139 G / A in the gene encoding MEH;
Glutamine 27 glutamic acid C / G in the gene encoding ADBR;
Polymorphisms that are in linkage disequilibrium with these polymorphisms;
20. The method of any one of claims 1-19, further comprising analysis of one or more polymorphisms selected from the group consisting of: The above analysis
-1607 1G / 2G (for the 2G allele only) within the promoter of the gene encoding MMP1;
-1562 C / T in the promoter of the gene encoding MMP9;
M1 null in the gene encoding GST-1;
1237 G / A in the 3 ′ region of the gene encoding α1-antitrypsin;
-82 A / G within the promoter of the gene encoding MMP12;
T → C within codon 10 of the gene encoding TGFβ;
760 C / G within the gene encoding SOD3;
-1296 T / C within the promoter of the gene encoding TIMP3;
S mutations in the gene encoding α1-antitrypsin; and polymorphisms that are in linkage disequilibrium with these polymorphisms;
40. The method of any one of claims 1-20 and 39, further comprising analysis of one or more polymorphisms selected from the group consisting of: A method to determine whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or a method of diagnosing the likelihood of developing COPD and / or emphysema And
Examining the presence or absence of at least one protective SNP genotype associated with reduced predisposition to COPD and / or emphysema, and / or reduced potential risk, and / or reduced incidence;
In the absence of any protective SNP genotype, examine the presence or absence of at least one susceptible SNP genotype associated with COPD and / or emphysema trends and / or potential risk and / or development Including operations;
The presence of one or more protective SNP genotypes indicates a decreased predisposition to COPD and / or emphysema and / or a reduced potential risk and / or a reduced incidence, and any protective SNP A method wherein the combination of absence of genotype and presence of at least one susceptible SNP genotype indicates an increased predisposition to COPD and / or emphysema and / or an increased risk and / or increased onset . Selection of the at least one protective SNP genotype,
-765 CC or CG within the promoter of the gene encoding COX2;
130 arginine / glutamine AA within the gene encoding IL13;
298 aspartic acid / glutamic acid TT within the gene encoding NOS3;
Lysine 420 threonine AA or AC within the gene encoding VDBP;
Glutamic acid 416 aspartic acid TT or TG in the gene encoding VDBP;
Isoleucine 105 valine AA in the gene encoding GSTP-1;
MS in the gene encoding α1-antitrypsin;
+489 GG in the gene encoding TNFα;
-308 GG in the gene encoding TNFα;
C89Y AA or AG within the gene encoding SMAD3;
161 GG within the gene encoding MBL2;
-1903 AA within the gene encoding CMA1;
Arginine 197 glutamine AA in the gene encoding NAT2;
Histidine 139 arginine GG in the gene encoding MEH;
-366 AA or AG within the gene encoding ALOX5;
HOM T2437C TT within the gene encoding HSP70;
Exon 1 +49 CT or TT within the gene encoding elafin;
Glutamine 27 glutamate GG in the gene encoding ADBR;
-1607 1G1G or 1G2G within the gene encoding MMP1;
23. The method of claim 22, wherein the method is performed from the group consisting of: Selection of the at least one protective SNP genotype,
+ 760GG or + 760CG in the gene encoding SOD3;
-1296TT within the promoter of the gene encoding TIMP3;
CC (homo P allele) within codon 10 of the gene encoding TGFβ;
24. The method of claim 23, wherein the method is also performed from a group consisting of: When testing the susceptibility SNP genotype,
The selection of the susceptibility SNP genotype,
105 AA in the gene encoding interleukin-18;
-133 CC in the promoter of the gene encoding interleukin-18;
-675 5G5G in the promoter of the gene encoding plasminogen activator inhibitor 1;
-1055 TT within the promoter of the gene encoding interleukin-13;
874 AA in the gene encoding interferon gamma;
+489 AA or AG within the gene encoding TNFα;
-308 AA or AG within the gene encoding TNFα;
C89Y GG in the gene encoding SMAD3;
E469K GG in the gene encoding ICAM1;
Glycine 881 arginine GC or CC within the gene encoding NOD2;
-511 GG in the gene encoding IL1B;
Tyrosine 113 histidine TT in the gene encoding MEH;
-366 GG within the gene encoding ALOX5;
HOM T2437C CC or CT within the gene encoding HSP70;
+13924 AA within the gene encoding CLCA1; and
-159 CC within the gene encoding CD-14;
The method according to any one of claims 22 to 24, wherein the method is carried out from the group consisting of When testing the susceptibility SNP genotype,
The selection of the susceptibility SNP genotype,
-82 AA within the promoter of the gene encoding MMP12;
-1607 2G2G within the promoter of the gene encoding MMP1;
-1562 CT or -1562 TT in the promoter of the gene encoding MMP9; and 1237 AG or 1237 AA in the 3 'region of the gene encoding α1-antitrypsin (the allelic genotype is Tt or tt);
26. The method of claim 25, wherein the method is also performed from the group consisting of:   One or more nucleotide probes and / or nucleotide primers for use in the method of any one of claims 1 to 26, wherein the one or more nucleotide probes and / or nucleotide primers are: Covers the polymorphic region in the gene in which the polymorphism to be analyzed exists, or there is a polymorphism to be analyzed in the gene using its one or more nucleotide probes and / or nucleotide primers Nucleotide probes and / or nucleotide primers that can be used to cover polymorphic regions.   A nucleic acid sequence capable of hybridizing to a nucleic acid sequence encoding one or more sensitive or protective polymorphisms selected from the group of claim 1 or to a complementary sequence thereof is presented. A nucleic acid microarray having a patterned substrate.   24. A method of treating a subject having an increased predisposition to and / or potential risk of developing COPD and / or emphysema, or a subject developing COPD and / or emphysema, selected from the group of claim 23 Reproducing the presence and / or functioning effect of the protected genotype with respect to genotype or phenotype in the subject's body.   A subject with an increased predisposition to and / or potential risk of developing COPD and / or emphysema, or a subject who has developed COPD and / or emphysema, and has a physiologically active concentration of the expressed gene product Wherein the subject has a detectable susceptibility genotype selected from the group of claim 25, wherein the expression of the gene is upregulated or downregulated to be outside the normal range for the subject's age and gender. A method of treatment comprising returning the physiologically active concentration of the expressed gene product to a range that is normal for the age and sex of the subject.   A subject with a predisposition to and / or a potential risk of developing COPD and / or emphysema, or a subject who has COPD and / or emphysema and is within the promoter of the gene encoding COX2 -765 C A method of treating a subject known to have a GG genotype at the position of the / G polymorphism, and administering to the subject an agent that can reduce COX2 activity in the subject's body A method involving operations.   32. The method of claim 31, wherein the agent is a COX2 inhibitor or a non-steroidal anti-inflammatory drug (NSAID).   33. The method of claim 32, wherein the selection of the COX2 inhibitor is performed from the group consisting of Celebrex (celecoxib), Bextra (valdecoxib), Biox (rofecoxib).   A subject with a predisposition to and / or a potential risk of developing COPD and / or emphysema, or a subject who has COPD and / or emphysema, and has an average of 105 C / A in the gene encoding interleukin-18 A method of treating a subject whose AA genotype is known to exist at the location of the mold, and administering an agent capable of increasing the activity of interleukin 18 in the subject's body Including methods.   -133 G / C in a gene that predisposes to and / or has a potential risk of developing COPD and / or pulmonary emphysema, or is COPD and / or pulmonary emphysema and encodes interleukin-18 A method of treating a subject whose CC genotype is known to be present at the polymorphic position, wherein the subject is administered a drug capable of increasing the activity of interleukin 18 in the subject's body A method involving operations.   Within the promoter of a gene encoding plasminogen activator inhibitor 1 that is a subject with a predisposition to and / or a potential risk of developing COPD and / or emphysema, or who has COPD and / or emphysema -675 Treatment of a subject known to have a 5G5G genotype at the position of the 4G / 5G polymorphism, which can increase the activity of plasminogen activator inhibitor 1 in the subject's body A method comprising administering an agent to the subject.   Subjects with a predisposition to and / or potential risk of developing COPD and / or pulmonary emphysema, or subjects with COPD and / or pulmonary emphysema, with 874 A / T in the gene encoding interferon-γ A method of treating a subject whose AA genotype is known to be present at the location of the mold, and administering an agent capable of altering interferon-γ activity in the subject's body Including methods.   -159 C / T in a gene that is predisposed to and / or a potential risk of developing COPD and / or emphysema, or who has COPD and / or emphysema, and that encodes CD-14 A method of treating a subject whose CC genotype is known to be present at the polymorphic position, wherein a drug capable of altering CD-14 and / or IgE activity in the subject's body A method comprising an operation of administering to a subject. A method to determine whether a subject has a predisposition to and / or a potential risk of developing chronic obstructive pulmonary disease (COPD) and / or emphysema, and / or a method of diagnosing the likelihood of developing COPD and / or emphysema And
-765 C / G in the promoter of the gene encoding cyclooxygenase (COX2);
105 C / A within the gene encoding interleukin 18 (IL18);
-133 G / C within the promoter of the gene encoding IL18;
-675 4G / 5G in the promoter of the gene encoding plasminogen activator inhibitor 1 (PAI-1);
874 A / T in the gene encoding interferon-γ (IFN-γ);
16 arginine / glycine in the gene encoding the β2 adrenergic receptor;
130 arginine / glutamine (G / A) within the gene encoding interleukin 13 (IL13);
298 aspartic acid / glutamic acid in the gene encoding NO synthase 3 (NOS3);
Isoleucine 105 valine (A / G) in the gene encoding glutathione S-transferase P (GST-P);
Glutamic acid 416 aspartic acid (T / G) in the gene encoding vitamin D binding protein (VDBP);
Lysine 420 threonine (A / C) in the gene encoding vitamin D binding protein;
-1055 C / T in the promoter of the gene encoding interleukin-13;
an S mutation in the gene encoding α1-antitrypsin;
+489 G / A in the gene encoding tumor necrosis factor α (TNFα);
C89Y A / G in the gene encoding SMAD3;
E469K A / G in the gene encoding intercellular adhesion molecule 1 (ICAM1);
Glycine 881 arginine G / C in the gene encoding caspase (NOD2);
161 G / A in the gene encoding mannose-binding lectin 2 (MBL2);
-1903 G / A in the gene encoding chymase 1 (CMA1);
Arginine 197 Glutamine G / A in the gene encoding N-acetyltransferase 2 (NAT2);
-366 G / A in the gene encoding 5 lipo-oxygenase (ALOX5);
HOM T2437C in the gene encoding heat shock protein 70 (HSP70);
+13924 T / A in the gene encoding calcium-dependent chloride channel 1 (CLCA1);
-159 C / T in the gene encoding monocyte differentiation antigen CD-14 (CD-14);
Exon 1 +49 C / T in the gene encoding elafin;
-308 G / A in the promoter of the gene encoding TNFα;
-511 A / G in the promoter of the gene encoding interleukin 1B (IL1B);
Tyrosine 113 histidine T / C in the gene encoding microsomal epoxide hydrolase (MEH);
Arginine 139 G / A in the gene encoding MEH;
Glutamine 27 glutamate C / G within the gene encoding ADBR; and
-1607 1G / 2G (for the 1G allele only) within the promoter of the gene encoding MMP1;
A method comprising analyzing two or more polymorphisms selected from the group consisting of:   2. An antibody microarray comprising a substrate presenting an antibody capable of binding to an expression product of a gene whose expression is upregulated or downregulated when associated with a sensitive or protective polymorphism selected from the group of claim 1. A method for screening for a compound that alters the expression and / or activity of a gene whose expression is up- or down-regulated when associated with a susceptibility or defensive polymorphism selected from the group of claim 1 comprising:
Contacting a candidate compound with a cell containing a sensitive or protective polymorphism selected from the group of claim 1 known to be associated with upregulation or downregulation of gene expression;
Measuring the expression of the gene after contact with the candidate compound,
A method wherein the change in expression level after the contacting step as compared to before the contacting step indicates the ability of the compound to alter the expression and / or activity of the gene.   42. The method of claim 41, wherein the cells are human lung cells whose presence of the polymorphism has been confirmed by preliminary screening.   43. The method of claim 42, wherein the cell contains a susceptibility polymorphism associated with downregulation of expression of the gene, and the screening is for screening candidate compounds that upregulate expression of the gene. .   43. The method of claim 42, wherein the cell contains a susceptibility polymorphism associated with upregulation of expression of the gene, and the screening is for screening candidate compounds that downregulate expression of the gene. .   43. The cell of claim 42, wherein the cell contains a protective polymorphism associated with upregulation of expression of the gene, and the screening is for screening candidate compounds that further upregulate expression of the gene. Method.   43. The cell of claim 42, wherein the cell contains a protective polymorphism associated with downregulation of the gene expression and the screening is for screening candidate compounds that further downregulate expression of the gene. Method. A method of screening for a compound that modulates the expression and / or activity of a gene whose expression is upregulated or downregulated when associated with a susceptibility or defensive polymorphism selected from the group of claim 1 comprising:
A cell comprising a gene whose expression is upregulated or downregulated when associated with a susceptibility or defense polymorphism selected from the group of claim 1 wherein the expression of the gene is upregulated or downregulated in the cell Contacting the candidate compound with (not regulated);
Measuring the expression of the gene after contacting with the candidate compound,
A method wherein the change in expression level after the contacting step as compared to before the contacting step indicates the ability of the compound to alter the expression and / or activity of the gene.   48. The method of claim 47, wherein the cells are human lung cells whose presence and expression baseline levels have been confirmed by preliminary screening.   49. The method of claim 48, wherein expression of the gene is downregulated when associated with a susceptibility polymorphism, and the screening is for screening candidate compounds that upregulate expression of the gene in the cell.   49. The method of claim 48, wherein expression of the gene is upregulated when associated with a susceptibility polymorphism, and the screening is for screening candidate compounds that downregulate the expression of the gene in the cell.   49. The method of claim 48, wherein expression of the gene is upregulated when associated with a defense polymorphism, and the screening is for screening candidate compounds that upregulate expression of the gene in the cell.   49. The method of claim 48, wherein expression of the gene is down-regulated when associated with a defense polymorphism, and the screening is for screening candidate compounds that down-regulate expression of the gene in the cell.   Prophylaxis, including manipulation of a subject with a predisposition to COPD or emphysema, or a subject diagnosed with COPD or emphysema, to return the physiologically active concentration of the expressed gene product to a range that is normal for the subject's age and gender A method for assessing the likelihood of responding to a method or therapy, wherein the presence or absence of a sensitive polymorphism selected from the group of claim 1 is detected and present in the body of the subject. The susceptibility polymorphism up-regulates or down-regulates the expression of the gene, so that the physiologically active concentration of the expressed gene product is outside the normal range for the age and sex of the subject. A method comprising detecting an existence, wherein the presence of the polymorphism is detected indicating that the subject is likely to respond to the treatment.

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