KR101668813B1 - Composition, kit, and microarray for diagnosing chronic obstructive pulmonary disease and method for diagnosis of chronic obstructive pulmonary disease using the same - Google Patents

Abstract

A kit, a microarray, and a COPD diagnosis method using the same. This can be used to accurately and sensitively diagnose COPD of an individual.

Description

TECHNICAL FIELD The present invention relates to a composition for the diagnosis of chronic obstructive pulmonary disease, a kit, a microarray, and a method for diagnosing chronic obstructive pulmonary disease using the composition, kit, and microarray for diagnosing chronic obstructive pulmonary disease.

A kit, a microarray, and a method for diagnosing chronic obstructive pulmonary disease using the same.

In chronic obstructive pulmonary disease (COPD), an abnormal inflammatory reaction occurs in the lungs due to the ingestion of harmful particles or gas, which leads to progressive restriction of airflow, resulting in decreased pulmonary function and respiratory distress Emphysema, chronic bronchitis, and so on. The main cause of COPD is smoking, but COPD can be induced by complex interactions of genetic and environmental factors as well as smoking. Genetic incentives are associated with genes, airway hyperresponsiveness, etc. Environmental factors include not only smoking but also occupational dust, chemicals, indoor and outdoor air pollution.

COPD is diagnosed by pulmonary function tests. Pulmonary function tests are based on spirometric measures and measure lung volume and lung capacity. The forced expiratory volume in 1 second (FEV ₁ ) and forced vital capacity (FVC) were measured by spirometry. FEV ₁ refers to the volume of 1 second after a maximum withdrawal, and FVC refers to the maximum amount of lung capacity an individual can artificially do. The ratio of FEV ₁ to FVC, ie, 1-second forced expiratory volume in one second, is used as an index to diagnose COPD.

Human genome-based studies have identified genetic factors associated with FEV ₁ or FVC. However, genetic factors associated with smoking, a leading cause of COPD or COPD, have not yet been identified.

Accordingly, there is a need to develop a composition, a kit, a microarray, and a COPD diagnosis method for diagnosing COPD through genetic analysis with high sensitivity and accuracy.

A composition for diagnosing COPD is provided.

Provides a COPD diagnostic kit.

A microarray for COPD diagnosis is provided.

Provides a COPD diagnostic method.

One aspect includes a probe or primer comprising a polynucleotide that is the same as or complementary to a polynucleotide comprising eight or more contiguous nucleotides comprising a 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2 A composition for the diagnosis of chronic obstructive pulmonary disease (COPD) is provided.

The polynucleotide of SEQ ID NO: 1 or 2 may be included in the ACN9 gene. The term "gene" means a unit for making each trait and may be a region of the genomic sequence corresponding to the unit of trait. The gene may comprise a regulatory region, a transcription region, or other functional sequence region. The functional sequence region may be an intron that is not transcribed. The ACN9 gene is a nucleic acid encoding the Acn9 protein involved in the gluconeogenesis of the grape. The ACN9 gene can be obtained, for example, from GenBank Accession No. < RTI ID = Nucleic acid of NC_000007.13, or a nucleic acid of GenBank Accession No. Or a nucleic acid encoding the amino acid sequence of NT_007933.16. The polynucleotide of SEQ ID NO: 1 or 2 may be contained in the intron of the ACN9 gene.

The position of the 26 < th > nucleotide from the 5 ' end of the polynucleotide of SEQ ID NO: 1 or 2 may be a single base polymorphic position. The term " single nucleotide polymorphism "(SNP) refers to the difference in single base-pair variation in DNA between an individual and an individual, and the most common form of DNA polymorphism / 1kb). The SNP position refers to a nucleotide representing a SNP in a polynucleotide sequence. The polynucleotide may be DNA or RNA. The nucleotide of the nucleotide at the SNP position in SEQ ID NO: 1 may be the allele adenine (A) or the allelic thymine (T). The nucleotide of the nucleotide at the SNP position in SEQ ID NO: 2 may be an allele cytosine (C) or an allele guanine (G). The term "allele" refers to a gene of alleles that can be paired. The allele may be a single nucleotide present at the same position.

The polynucleotide may comprise at least 8, at least 10, at least 15, at least 20, at least 30, or at least 40 consecutive nucleotides comprising the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2, May be the same or complementary.

The probe refers to an oligonucleotide that specifically binds to a target sequence. Probes include polynucleotides that include specific portions designed to hybridize in a sequence-specific manner, for example, with complementary regions of a specific nucleic acid sequence, such as a target nucleic acid sequence. The probe may comprise DNA, RNA, or a combination thereof, and may be labeled with a detectable label. The detectable label may be a fluorescent dye compound bound to the probe by covalent or non-covalent bonds, for example a FRET (Forter Resonance Energy Transfer) pair of a fluorescent donor and a fluorescent acceptor. The probe is an allele-specific probe in which a polymorphic site is present in a nucleic acid fragment and hybridizes to a nucleic acid fragment containing an allele but not to a nucleic acid fragment containing another allele.

The primer may be an oligonucleotide that provides a polymerization initiation point in a polymerization reaction by a polymerase, and the primer may be used in a nucleic acid amplification reaction. The primer is hybridized with a region complementary to the target sequence. The term "amplification" refers to increasing the number of copies of the target sequence or its complementary sequence. The nucleic acid amplification reaction can be performed by a method known in the art. Amplification of nucleic acids involves either a method requiring multiple cycles during amplification or a method performed at a single temperature. Examples of cycling techniques include those that require thermal cycling. Methods that require thermocycling include polymerase chain reaction (PCR). PCR is known in the art. Isothermal amplification methods include, but are not limited to, strand displacement amplification (SDA), helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification RPA), loop mediated amplification (LAMP), nucleic acid based amplification (NASBA and TMA), or a combination thereof. The primers may be included in one, or two or more sets depending on the amplification method selected. The primer may be an allele-specific primer. If the product is amplified by amplification, the specific allele may be present.

Chronic obstructive pulmonary disease (COPD) includes emphysema, chronic bronchitis, or a combination thereof.

Another aspect is a probe or primer comprising a polynucleotide that is the same as or complementary to a polynucleotide comprising eight or more consecutive nucleotides comprising a 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2; And a reagent necessary for the polymerization reaction.

The polynucleotide, probe, primer, and COPD comprising 8 or more consecutive nucleotides comprising the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2 are as described above.

The kit may further comprise reagents necessary for the polymerization reaction. The reagents may include dNTPs, polymerases, buffers, indicators, or combinations thereof.

Another aspect provides a microarray for COPD diagnosis comprising a polynucleotide that is the same as or complementary to a polynucleotide comprising 8 or more contiguous nucleotides comprising the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2 do.

A polynucleotide comprising 8 or more consecutive nucleotides comprising the 26 th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2; Primer, and COPD are as described above.

The microarray may be one in which the polynucleotide is immobilized on a substrate. The polynucleotide may be immobilized on a substrate coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde. In addition, the substrate may be selected from the group consisting of a silicon wafer, glass, quartz, metal, and plastic. As the method of immobilizing the polynucleotide on the substrate, a micropipetting method using a piezoelectric method or a method using a pin-type spotter can be used.

Another aspect is a method comprising: obtaining a nucleic acid sample from a biological sample isolated from an individual; And determining the genotype of the 26th nucleotide from the 5 'end of the polynucleotide of SEQ ID NO: 1 or 2 from the obtained nucleic acid sample.

The polynucleotide and the COPD comprising 8 or more consecutive nucleotides including the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2 are as described above.

The method comprises obtaining a nucleic acid sample from a biological sample isolated from an individual.

The subject may be a mammal. The mammal may be a primate. The mammal may be a human, mouse, cow, pig, horse, sheep, dog, cat, or a combination thereof. The subject may be a subject suspected of having COPD. The subject may be a smoker. The smoker includes a person who smokes or indirectly exposed to tobacco smoke. The smoker may be an individual having a lower third level of the smoking intensity. For example, the lower third of the smoking intensity may be 35 years (pack-year; PY), less than 35 years, 1 to 35 years, 10 to 35 years, 20 to 35 years, or 30 to 35 years have. The number of cigarettes consumed per year is estimated based on the number of cigarettes smoked per capita per year for one year, and the number of cigarettes per day and the number of years smoked can be calculated.

The biological sample may be a solid tissue, such as a fresh or preserved organ or tissue sample or biopsy derived from an individual; Blood or blood components; Bodily fluids such as amniotic fluid, peritoneal fluid, or interstitial fluid; Cell, or a combination thereof. The sample may include a compound that is not naturally mixed with biological materials such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. The biological sample may be, for example, lung tissue. Lung tissue can be obtained by surgical procedures, or by optical extraction through material or laser. The amount of the transcript of the ACN9 gene comprising the polynucleotide of SEQ ID NO: 1 or 2 can be reduced compared to the control. The control group may be derived from a normal person who does not suffer from COPD disease as a negative control.

The nucleic acid sample may be a sample containing a nucleic acid. The nucleic acid may be a nucleic acid separated or purified from the biological sample. Methods for separating or purifying nucleic acids from biological samples may be those known in the art.

The method includes determining the genotype of the 26th nucleotide from the 5 'end of the polynucleotide of SEQ ID NO: 1 or 2 from the obtained nucleic acid sample.

The term "genotype" refers to the genetic composition and may be the presence of an allele or an allele.

The determination of the genotype can be carried out by various methods known in the art. The determination of the genotype may be accomplished by, for example, allele-specific probe hybridization, allele-specific amplification, sequencing, 5 'nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism, or a combination thereof Lt; / RTI >

The method may further comprise determining as COPD when the allele of the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 is thymine (T).

The method may further comprise determining the polynucleotide of SEQ ID NO: 2 as COPD when the allele of the 26th nucleotide from the 5 'terminus is guanine (G).

Another aspect is a method comprising: obtaining a nucleic acid sample from a biological sample isolated from an individual; And determining the genotype of the 26th nucleotide from the 5 'end of the polynucleotide of SEQ ID NO: 1 or 2 from the obtained nucleic acid sample. The information required for the COPD diagnosis of an individual may be information on lung function.

According to a composition for COPD diagnosis, a kit, a microarray, and a COPD diagnosis method using the same, the COPD of an individual can be used for accurate and sensitive diagnosis.

FIG. 1 is a graph showing gene expression levels according to the genotype of ACN9 (y axis: FPKM).

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

Example  One. COPD  Identification of differentially expressed genes in lung tissue of patients

1.1. Screening of subjects

1,000 COPD patients and 1,000 normal people were studied. COPD patients were recruited from lung clinics in 24 hospitals belonging to the Korean obstructive lung disease (KOLD) study group or the ASAN COPD network. The control group, normal persons, were recruited from the patients who received health screening at Asan Medical Center (Seoul, Korea). All normal people are currently smoking or have experienced smoking. Subjects were tested for pulmonary function according to the American Thoracic Society / European Respiratory Society criteria. Normal people had normal pulmonary function, whereas normal COPD patients had a FEV ₁ / FVC of less than 0.7 after bronchodilator administration. The amount of tobacco consumption based on the year of smoking (pack-year; PY), ie, one cigarette per day for one year, was calculated as the number of cigarettes per day and the number of years of smoking. All subjects agreed in writing and each study protocol was approved by the Clinical Trials Committee.

Basic information of the subject is shown in Table 1 below.

Characteristic Patients with COPD (n = 1,000) Normal subjects (n = 1,000) p value male(%) 977 people (97.7%) 983 (98.3%) 0.42 Age (mean ± SD) 69.2 ± 7.8 57.2 ± 9.5 <0.0001 Smoking, PY (mean ± SD) 44.9 ± 23.1 34.7 ± 27.8 <0.0001 FEV ₁ (L) 1.64 ± 0.61 3.41 ± 0.68 <0.0001 FEV ₁ , predicted% 55.9 ± 18.6 95.9 ± 32.6 <0.0001 FEV ₁ / FVC 48.9 ± 12.1 78.5 ± 22.8 <0.0001

* SD (standard deviation); Standard Deviation

In Table 1, FEV ₁ (L) refers to forced expiratory volume in 1 second (FEV ₁ ) and FVC refers to forced vital capacity (FVC). FEV ₁ / FVC refers to% forced expiratory volume in one second.

As shown in Table 1, COPD patients were older than normal controls and had a higher number of tobacco babies. In addition, COPD patients had lower pulmonary function than normal subjects.

1.2. Selection of target gene and genotype analysis

Thirteen single nucleotide polymorphisms (SNPs) were selected based on the meta-analysis of the genome wide association study (GWAS) results of the Korean population cohort. SNPs were genotyped on an Applied Biosystems 7900HT real-time PCR system using TaqMan technology (Applied Biosystems, Foster City, Calif.).

Of the 13 SNPs, the alleles of ACN9, which seem to be related to COPD, are shown in Table 2 below.

SNP
(Reference SNP cluster number) Chromosome number location gene Reference
Allele Alternative
Allele MAF Coefficient
(beta) rs10231916 7 96763456 ACN9 A T 0.51 - rs10229181 7 96795041 ACN9 C G 0.51 -

In Table 2, the reference allele refers to the reference allele, and the alternative allele refers to alleles mutated into polymorphisms. The minor-allele frequency (MAF) represents the allele frequency. The nucleic acid sequence of ACN9 is GeneBank Accession No. &lt; RTI ID = 0.0 &gt; Nucleotide sequence of NC_000007.

According to the dbSNP138 database (https://bioq.saclab.net/query/submit.php?db=bioq_dbsnp_human_138), the SNN of ACN9 is located in the intron of the ACN9 gene as a mutation of the 96763456th nucleotide from the 5 'end of chromosome 7 , And the base of the nucleic acid is an adenine (A) or a thymine (T). rs10231916 is the polymorphism of the 26 &lt; th &gt; nucleotide from the 5 &apos; end in the nucleotide sequence of SEQ ID NO: 1 below and is indicated in bold and parentheses.

rs10231916: 5'-GCAGCAGATGATACAGCAAACACGT [A / T] ACTGCAAAAACAGCTACAGCAGCTA-3 '(SEQ ID NO: 1)

The SNP of ACN9, rs10229181, is located in the intron of the ACN9 gene as a mutation of nucleotide 96795041 from the 5 'end of chromosome 7 according to the dbSNP138 database, and has a polymorphism in which the nucleotide base is cytosine (C) or guanine (G). rs10229181 is the polymorphism of the 26th nucleotide from the 5 ' end in the nucleic acid sequence of SEQ ID NO: 2 below and is indicated in bold and parentheses.

rs10229181: 5'-GAAACTGAAGAGGTTAATATGCAGA [C / G] TGCAAGGCTAAGTTGGCAGTTTGCC-3 '(SEQ ID NO: 2)

1.3. Genetic association analysis

The Hardy-Weinberg equilibrium was evaluated using a fitness test. Estimating additional genetic models and using SAS ver. 9.2 (SAS Inc., Cary, NC, USA) program used logistic regression analysis to adjust for age, sex, and smoking PY. In addition to the whole model, models including the SNP-by-pack-years interaction term (SNP) and the smoking intensity-stratified model were evaluated. Linear regression analysis showed that FEV ₁ and FEV ₁ as quantitative phenotypes after age, sex, To-FVC ratio.

COPD sensitivity and genetic linkage analysis, and correlations with cigarette smoking were shown in Table 3 below.

SNP
(Reference SNP cluster number) Chromosome number gene Genotype COPD (persons) Control group (persons) OR
95% CI
P _SNP P _{i nt} rs10231916 7 ACN9 AA
AT
TT 271
494
232 248
549
203 1.043
(0.893-1.219)
0.60 1.3x10 ^-8 rs10229181 7 ACN9 CC
CG
GG 265
479
236 245
544
204 1.064
(0.910-1.243)
0.44 8.1x10 ^-9

In Table 3, OR indicates the odd ratio, CI indicates the confidence interval, P _SNP indicates the p-value of the association analysis of COPD sensitivity and SNP, P _int indicates COPD sensitivity, Is the p-value of the interrelation.

As shown in Table 3, the SNP rs10231916 and rs10229181 of ACN9 showed a normal deviation from the Hardy-Weinberg equilibrium in the control group, but not in the whole group, and showed a significant correlation with COPD disease and smoking. In Table 3, if P _{i nt} is less than 0.05, there is a significant interaction between SNP and smoking. Thus, SNPs rs10231916 and rs10229181 of ACN9 were associated with P _{i nt,} and interaction was significant.

In addition, a genetic association analysis according to the smoking intensity was performed in three quintiles according to the smoking intensity of COPD patients, and the results are shown in Table 4 below. The criteria for the third quintile were divided into the lower third quintile in the under 35 years, the third quintile in the middle years and the upper third quintile in the more than 50 years.

SNP
(Reference SNP cluster number) Chromosome number gene Lower third quartile Middle third Top third quartile rs10231916 7 ACN9 1.25 (1.00-1.56), 0.051 0.97 (0.78-1.20), 0.77 0.82 (0.64-1.06), 0.13 rs10229181 7 ACN9 1.29 (1.03-1.61), 0.028 1.00 (0.81-1.24), 0.98 0.82, (0.64-1.06), 0.13

In Table 4, the values represent multiplication ratios (confidence intervals), and p values.

If the odds ratio is greater than 1, the risk of COPD disease is increased. If the odds ratio is less than 1, the risk of COPD disease is lowered. Only if the confidence interval does not include 1, it significantly reduces or increases the risk. Also, p <0.05 indicates a significant association between SNP and COPD disease. As shown in Table 4, COPD disease and rs10229181 were significantly correlated only in the lower third quintile of smoking.

On the other hand, the relationship between lung function and SNP is shown in Table 5 below.

SNP
(Reference SNP cluster number)
All subjects (n = 2,000) Patients with COPD (n = 1,000) FEV ₁ FEV ₁ / FVC FEV ₁ FEV ₁ / FVC rs10231916 -0.028 (0.027)
0.30 -1.460 (0.712)
0.04 -0.018 (0.026)
0.49 -0.496 (0.547)
0.37 rs10229181 -0.028 (0.027)
0.04 -1.524 (0.716)
0.03 -0.007 (0.026)
0.78 -0.427 (0.550)
0.44

In Table 5, the values represent the beta (error) value and the p value. The β value is interpreted as indicating directionality, so a negative β value is interpreted to be associated with lung function deterioration. Also, p <0.05 indicates a significant association between SNP and pulmonary function.

As shown in Table 5, the 'T' allele of rs10231916 in ACN9 was associated with low FEV ₁ / FVC values, consistent with the results of the whole population. In addition, it was confirmed that there was a significant correlation between lung function and rs10231916 and rs10229181 of ACN 9.

1.4. Gene expression in lung tissue

Gene expression profiling data of ACN9 were obtained by RNA sequencing using lung tissues from 193 COPD patients or normal individuals.

Specifically, for RNA sequencing, RNA was extracted from lung tissue obtained using the RNeasy 96 Universal tissue kit (Qiagen, Gaithersburg, MD, USA). Qualitative and quantitative determination of the total RNA obtained was performed spectrophotometrically using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and electrophoretically confirmed using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, Calif., USA) Respectively. To construct a library compatible with Illumina, the TruSeq RNA library preparation kit (Illumina, San Diego, CA, USA) was used according to the manufacturer's instructions. Briefly, purified messenger RNA from total RNA was chemically fragmented using polyA sorting and converted to single stranded cDNA using random hexamer priming. Double-stranded (ds) cDNA was generated to construct the TruSeq library. Short ds-cDNA fragments were ligated to the sequencing adapter and appropriate fragments were separated by agarose gel electrophoresis. The TruSeq RNA library constructed by PCR amplification was quantitated using qPCR according to the quantitative PCR (qPCR) quantitation protocol guide and the amount was electrophoretically quantified using Bioanalyzer 2100 (Agilent Technologies). Nucleotide sequence analysis was performed using the HiSeq (TM) 2000 platform (Illumina).

The level of fragments per kilobase of exon per million fragments mapped (FPKM) mapped per million pieces of mRNA was analyzed using a t- test to confirm differential expression according to the genotype of the candidate gene. FPKM represents the level of expression.

To investigate whether gene mutations affect gene expression, genotypes of ACN9 were analyzed in 93 individuals who could obtain blood samples, regardless of disease status. Linear regression analysis was performed to investigate the relationship between expression level and genotype.

The expression of ACN9 is shown in Table 6 below, and the gene expression level according to the genotype of ACN9 is shown in Fig. 1 (fc: fold change).

gene COPD / normal fc COPD / normal standard deviation fc Normal average COPD average Normal standard deviation COPD. Standard Deviation COPD / normal P value of COPD / normal ACN9 -1.24344 1.60x10 ^-1 6.90x10 ^-1 5.95x10 ^-1 8.49x10 ^-2 1.35x10 ^-1 -5.85 2.59x10 ^-8

As shown in Table 6, there was a significant decrease in lung tissue of COPD patients ( p = 2.59x10 ^-8 ). Also, as shown in Fig. 1, the 'T' allele of rs10231916 in ACN9 tended to be lowered in 93 samples ( p = 0.07).

<110> Kangwon National University Hospital <120> Composition, kit, and microarray for diagnosing chronic          obstructive pulmonary disease          obstructive pulmonary disease using the same <130> PN107129 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> rs10231916 as a Single Nucleotide Polymorphism of ACN9 <220> <221> mutation (26) <223> Single nucleotide polymorphism from adenine (A) to thymine (T) <400> 1 gcagcagatg atacagcaaa cacgtaactg caaaaacagc tacagcagct a 51 <210> 2 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> rs10229181 <220> <221> mutation (26) <223> Single nucleotide polymorphism from cytosine (C) to guanine (G) <400> 2 gaaactgaag aggttaatat gcagactgca aggctaagtt ggcagtttgc c 51

Claims (14)

Comprising a probe or primer comprising a polynucleotide that is the same as or complementary to a polynucleotide comprising at least 8 consecutive nucleotides from the 5 'end to the polynucleotide of SEQ ID NO: 1 or 2, A composition for the diagnosis of chronic obstructive pulmonary disease (COPD)
Wherein the polynucleotide of SEQ ID NO: 1 or 2 is contained in the ACN9 gene. delete The composition for diagnosing COPD according to claim 1, wherein the position of the 26th nucleotide from the 5 'end of the polynucleotide of SEQ ID NO: 1 or 2 is a single nucleotide polymorphism (SNP) position. 4. The composition for diagnosing COPD according to claim 3, wherein the nucleotide of the nucleotide at the SNP position in SEQ ID NO: 1 is the allele adenine (A) or the allele thymine (T). 4. The composition for diagnosing COPD according to claim 3, wherein the nucleotide of the nucleotide at the SNP position in SEQ ID NO: 2 is allele cytosine (C) or allele guanine (G). A probe or primer comprising a polynucleotide which is the same as or complementary to a polynucleotide comprising 8 or more consecutive nucleotides comprising the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2; And
A kit for the diagnosis of COPD comprising a reagent necessary for a polymerization reaction,
Wherein the polynucleotide of SEQ ID NO: 1 or 2 is contained in the ACN9 gene. 1. A microarray for COPD diagnosis comprising a polynucleotide that is the same as or complementary to a polynucleotide comprising 8 or more contiguous nucleotides comprising a 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 or 2,
Wherein the polynucleotide of SEQ ID NO: 1 or 2 is contained in the ACN9 gene. Obtaining a nucleic acid sample from the biological sample separated from the subject; And
Determining the genotype of the 26th nucleotide from the 5 'end of the polynucleotide of SEQ ID NO: 1 or 2 from the obtained nucleic acid sample,
Wherein the polynucleotide of SEQ ID NO: 1 or 2 is contained in the ACN9 gene. 9. The method of claim 8, further comprising determining as COPD when the allele of the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 1 is thymine (T). 9. The method according to claim 8, further comprising the step of determining COPD when the allele of the 26th nucleotide from the 5 'end in the polynucleotide of SEQ ID NO: 2 is guanine (G). 9. The method of claim 8, wherein the subject is a smoker. 12. The method according to claim 11, wherein the smoker is an individual having a smoking intensity lower third quintile. 13. The method of claim 12, wherein the smoking intensity lower quintile is less than 35 years old (pack-year) or 35 years old. The method according to claim 8, wherein the step of determining the genotype comprises at least one of allele-specific probe hybridization, allele-specific amplification, sequencing, 5'-nuclease digestion (5 'nuclease digestion), molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism, Or a combination thereof.

Images