chronic obstructive pulmonary disease ( copd ) is a disease characterized by airflow limitation that is not fully reversible ( 1 ) .
copd is a major health problem worldwide , with a high rate of occurrence , and high costs with its management .
it is the fourth leading cause of death in the world and the economic burdens associated with copd is ever increasing ( 1 , 2 ) .
inhaled cigarette smoke induces lung inflammation by various inflammatory cells , such as neutrophil , macrophage , and cd4 + t cells .
a variety of inflammatory mediators , such as protease , matrix metalloproteinase and tumor necrosis factor - alpha , can enhance lung parenchymal destruction , and cause airway remodeling .
these inflammatory processes can result airway narrowing and airflow limitation , finally develops copd ( 1 , 3 ) .
however , only a small fraction of smokers develops symptomatic copd , suggesting that genetic factors also contribute to the development of copd ( 4 - 6 ) .
recently , genome - wide association ( gwa ) studies have identified hundreds of genetic variants influencing the risk of complex human diseases , including copd ( 7 - 9 ) .
in addition , these gwa studies have aided our understanding of diverse molecular pathways underlying specific human diseases ( 10 , 11 ) .
the 15q25 region that contains the nicotinic acetylcholine receptor alpha subunit 5 and 3 ( chrna5 and chrna3 ) genes was initially identified as a lung cancer susceptibility locus through gwa studies conducted in caucasian populations ( 12 , 13 ) .
subsequently , this 15q25 region has been reported to be also associated with risk of copd in gwa studies ( 7 , 14 ) .
however , the single nucleotide polymorphisms ( snps ) , rs8034191 and rs1051730 , identified in previous gwa studies for lung cancer and copd in caucasians ( 7 - 14 ) , are extremely rare in asians , according to hapmap data .
( 15 ) reported that these two snps are very rare ( minor allele frequencies < 0.05 ) in chinese populations .
notably , they reported that among the snps in the chrna5-chrna3 locus , the rs6495309c > t in the promoter of the chrna3 gene affects the binding ability of the transcriptional factor , oct-1 , resulting in alteration of chrna3 expression , thereby influencing lung cancer risk ( 15 ) .
this finding suggests that the rs6495309c > t of chrna3 may be associated with the risk of copd in asian populations .
therefore , to verify the role of the rs6495309c > t of chrna3 on the risk of copd , we have carried out a case - control study in a korean population .
the patient group ( n = 406 ) consisted of male patients who visited the respiratory center in kyungpook national university hospital ( daegu , korea ) between july 2006 and december 2008 . diagnosis of copd was established by the nhlbi / who global initiative for copd ( gold ) ( 1 ) .
the criteria for copd were as follows : chronic respiratory symptoms and signs such as cough and dyspnea ; post - bronchodilator forced expiratory volume at one second ( fev1 ) < 80% of the predicted value , fev1/forced vital capacity ( fvc ) < 70% and fev1 reversibility after inhaling 200 g salbutamol < 12% of the pre - bronchodilator fev1 .
the severity of copd was classified by the guidelines of gold in terms of the percentage predicted fev1 : mild ( > 80% ) , moderate ( 50%-80% ) , severe ( 30%-50% ) or very severe ( < 30% ) . control subjects ( n = 394 ) were selected from a pool of healthy men who visited the general health check - up center .
the enrollment criteria for the control subjects were as follows : male , age > 45 yr , current or former smoker , no known disease and no history of any disease , and no airflow limitation .
all of the cases and controls were ethnic koreans that resided in daegu city , or in the surrounding regions .
genotypes of the rs6495309c > t was determined by melting - curve analysis using fluorescence labeled hybridization probes ( lightcycler , roche diagnostic , mannheim , germany ) . a genotype success rate of greater than 99% was achieved using the lightcycler .
samples that could not be scored by the lightcycler were re - genotyped by direct sequencing using an abi prism 3700 genetic analyzer ( applied biosystems , foster city , ca , usa ) .
all genotyping analyses were performed " blind " with respect to the case / control status to ensure quality control .
approximately 10% of the samples were randomly selected to be genotyped again by a different investigator , and the results were 100% concordant with the original analysis .
the cases and controls were compared using the student 's t - test for continuous variables , and a chi - squared test for categorical variables .
deviations of genotype frequencies among controls from those expected under hardy - weinberg equilibrium were assessed by chi - squared test ( 1 of freedom ) .
unconditional logistic regression analyses were used to calculate the odds ratio ( or ) and 95% confidence interval ( ci ) , with adjustment for possible confounders ( age , and pack - years of smoking used as continuous variables ) .
in addition to the overall association analysis , we performed a stratified analysis according to age , smoking status , and severity of copd to explore the association between genotypes and the risk of copd in each stratum .
a homogeneity test was performed in order to compare the difference between the genotype - related ors of the different groups .
all analyses were performed using statistical analysis system for windows , version 9.1 ( sas institute , cary , nc , usa ) .
this study was approved by the institutional review board of the kyungpook national university hospital ( approval no . , knuhbio_10 - 1015 ) .
the patient group ( n = 406 ) consisted of male patients who visited the respiratory center in kyungpook national university hospital ( daegu , korea ) between july 2006 and december 2008 . diagnosis of copd was established by the nhlbi / who global initiative for copd ( gold ) ( 1 ) .
the criteria for copd were as follows : chronic respiratory symptoms and signs such as cough and dyspnea ; post - bronchodilator forced expiratory volume at one second ( fev1 ) < 80% of the predicted value , fev1/forced vital capacity ( fvc ) < 70% and fev1 reversibility after inhaling 200 g salbutamol < 12% of the pre - bronchodilator fev1 .
the severity of copd was classified by the guidelines of gold in terms of the percentage predicted fev1 : mild ( > 80% ) , moderate ( 50%-80% ) , severe ( 30%-50% ) or very severe ( < 30% ) . control subjects ( n = 394 ) were selected from a pool of healthy men who visited the general health check - up center .
the enrollment criteria for the control subjects were as follows : male , age > 45 yr , current or former smoker , no known disease and no history of any disease , and no airflow limitation .
all of the cases and controls were ethnic koreans that resided in daegu city , or in the surrounding regions .
genotypes of the rs6495309c > t was determined by melting - curve analysis using fluorescence labeled hybridization probes ( lightcycler , roche diagnostic , mannheim , germany ) . a genotype success rate of greater than 99% was achieved using the lightcycler .
samples that could not be scored by the lightcycler were re - genotyped by direct sequencing using an abi prism 3700 genetic analyzer ( applied biosystems , foster city , ca , usa ) .
all genotyping analyses were performed " blind " with respect to the case / control status to ensure quality control .
approximately 10% of the samples were randomly selected to be genotyped again by a different investigator , and the results were 100% concordant with the original analysis .
the cases and controls were compared using the student 's t - test for continuous variables , and a chi - squared test for categorical variables .
deviations of genotype frequencies among controls from those expected under hardy - weinberg equilibrium were assessed by chi - squared test ( 1 of freedom ) .
unconditional logistic regression analyses were used to calculate the odds ratio ( or ) and 95% confidence interval ( ci ) , with adjustment for possible confounders ( age , and pack - years of smoking used as continuous variables ) .
in addition to the overall association analysis , we performed a stratified analysis according to age , smoking status , and severity of copd to explore the association between genotypes and the risk of copd in each stratum .
a homogeneity test was performed in order to compare the difference between the genotype - related ors of the different groups .
all analyses were performed using statistical analysis system for windows , version 9.1 ( sas institute , cary , nc , usa ) .
this study was approved by the institutional review board of the kyungpook national university hospital ( approval no . , knuhbio_10 - 1015 ) . written informed consent
age and pack - years of smoking were significantly higher in cases than in controls ( 64.4  7.5 vs 61.9  7.9 yr , p < 0.001 ; and 43.8  21.5 vs 31.7  16.4 pack - yr of smoking , p < 0.001 , respectively ) .
the fev1 and the fev1/fvc ratio were significantly lower in the copd group than in the control group ( p < 0.001 , both ) .
table 2 shows the genotype distribution of rs6495309c > t among the cases and controls .
the genotype distribution of the rs6495309c > t among the controls was in hardy - weinberg equilibrium .
the frequency of the variant t allele among the controls was 0.494 , which was comparable with that ( 0.482 ) of healthy chineses ( 15 ) .
the distribution of the rs6495309c > t genotypes was significantly different between the cases and controls ( cc , ct , and tt genotypes ; 34.5% , 48.8% , and 16.7% , respectively vs 26.6% , 48.0% , and 25.4% , respectively ; p = 0.004 ) , with the frequency of the variant t allele being significantly lower in the cases than in the controls ( 0.411 vs 0.494 , p = 0.001 ) .
the risk of copd decreased as the number of t alleles increased ( ptrend = 0.002 ) , and the tt genotype was associated with a significantly decreased risk of copd when compared to the cc genotype ( adjusted or , 0.50 ; 95% ci , 0.33 - 0.76 ; p = 0.001 ) .
individuals with the rs6495309 ct or tt genotype had a 31% ( 95% ci , 0.50 - 0.95 ; p = 0.023 ) decreased risk of copd when compared to those with the rs6495309 cc genotype .
the association between the rs6495309c > t genotypes and the risk of copd was further investigated , after stratification according to age , smoking status , and severity of copd ( table 3 ) .
the effects of the rs6495309c > t genotype on the risk of copd did not differ significantly between younger- and older - individuals , and former- and current - smokers ( p value of test for homogeneity [ ph ] , both , > 0.05 in all comparisons ) . when stratified by the median pack - years of smoking , the effect of the rs6495309c > t genotype on the risk of copd was more evident in lighter - smokers than in heavier - smokers under dominant model for the variant t allele ( ph = 0.030 ) .
when copd cases were categorized by disease severity , the rs6495309 ct , or tt genotype was associated with a significantly decreased risk of moderate to very severe copd ( gold ii - iv ; adjusted or , 0.56 ; 95% ci , 0.39 - 0.80 ; p = 0.001 ) , whereas there was no significant association between the rs6495309c > t genotypes and the risk of mild copd ( gold i ; adjusted or , 1.07 ; 95% ci , 0.69 - 1.65 ; ph = 0.024 ) .
in this study we determined the association between the rs6495309c > t polymorphism on the 15q25 region and the risk of copd in a korean population .
this finding was in agreement with results of a previous gwa study which was carried out in caucasians ( 7 ) in which the 15q25 region was a copd susceptibility locus , thus providing strong evidence that the 15q25 region may play an important role in the development of copd .
this study is the first report of an association between the chran3 rs6495309c > t and the risk of copd in asians .
acetylcholine receptors are classified into nicotinic acetylcholine receptors ( nachrs ) and muscarinic receptors , and nachrs are historically classified as neuronal- or muscle - type based on their initial site of identification and composite subunits ( 16 ) .
cholinergic parasympathetic activity in the airways induces tracheobronchial smooth muscle contraction and mucous secretion ( 17 , 18 ) . however , there is increasing evidence that non - neuronal cholinergic systems that are found in non - neuronal cells , such as airway inflammatory cells , modulate inflammation of the lung , and thus , contribute to the development of copd ( 19 ) .
it has been shown that bronchial epithelial cells , and airway fibroblasts and inflammatory cells express nachrs ( 20 ) .
nicotine and nicotine - derived nitrosamines in cigarette smoke triggers the nachrs of inflammatory cells of the lung to release protease and oxidants that are associated with the pathogenesis of copd ( 19 - 21 ) .
in addition , it has been demonstrated that the rs6495309 t allele significantly reduces promoter activity , which in turn diminishes chrna3 expression ( 15 ) .
therefore , it is plausible that subjects with a lower production of rs6495309 t allele for chrna3 might have a lower inflammatory response to smoking exposure , thus having a decreased susceptibility to copd .
the association between the chrna3 rs6495309c > t and risk of copd may be , in part , resulted from the fact that the snp affects nicotinic dependence .
it is known that nachrs expressed in the key regions of the brain play an important role in controlling smoking behavior such as nicotine dependence ( 22 - 25 ) .
several studies have reported that genetic variants of chromosome 15q25 , including the chrna3 rs6495309c > t , are responsible for tobacco nicotine dependence ( 15 , 25 ) . in the present study , however , there were no significant differences in genotype and allele frequencies according to smoking status and smoking exposure levels ( data not shown ) .
although the rs6495309 in the promoter of the chrna3 gene has been reported to be a functional snp , the association of the rs6495309 with copd may be due to linkage disequilibrium ( ld ) with other functional variant(s ) rather than a direct effect of the rs6495309 .
in order to identify the variant(s ) that may be in strong ld with the rs6495309 that could be actually responsible for the alteration in copd risk , we screened and estimated the lds of all the potentially functional snps ( with a minor allele frequency  0.05 in asians ) around the chrna5/chrna3 locus including the rs6495309 using the niehs database ( http://manticore.niehs.nih.gov ) .
as shown in fig . 1 , four snps ( rs578776 , rs472054 , rs660652 and rs615470 ) were captured .
however , none of these four snps were in strong ld with the rs6495309 based on hapmap jpt data of the public database ( http://www.ncbi.nlm.nih.gov/snp ) .
this finding suggests that the association observed in the present study may be due to direct effect of the rs6495309 .
however , because the four potentially functional snps captured from the database may also affect the risk of copd , future studies of these four snps are also needed to further understand the role of genetic variants in the chrna5/chrna3 locus in determining the risk of copd . in conclusion , this case - control study demonstrates a significant association of the chrna3 rs6495309c > t on chromosome 15q25 with the risk of copd . because this is the first case - control study investigating the association of the chrna3 rs6495309c > t with the risk of copd in asian populations , additional studies are required to confirm our findings .
 a genome - wide association study has identified the 15q25 region as being associated with the risk of chronic obstructive pulmonary disease ( copd ) in caucasians . 
 this study intended as a confirmatory assessment of this association in a korean population . 
 the rs6495309c > t polymorphism in the promoter of nicotinic acetylcholine receptor alpha subunit 3 ( chrna3 ) gene was investigated in a case - control study that consisted of 406 patients with copd and 394 healthy control subjects . 
 the rs6495309 ct or tt genotype was associated with a significantly decreased risk of copd when compared to the rs6495309 cc genotype ( adjusted odds ratio = 0.69 , 95% confidence interval = 0.50 - 0.95 , p = 0.023 ) . 
 the effect of the rs6495309c > t on the risk of copd was more evident in moderate to very severe copd than in mild copd under a dominant model for the variant t allele ( p = 0.024 for homogeneity ) . 
 the chrna3 rs6495309c > t polymorphism on chromosome 15q25 is associated with the risk of copd in a korean population . 
INTRODUCTION
MATERIALS AND METHODS
Study population
Genotyping
Statistical analysis
Ethic statement
RESULTS
DISCUSSION