In the current study, we observed that two common variants in the nAChR
locus on chromosome 15q24/25 affect FEV1
/FVC decline in a population-based sample consisting of heavy smokers (COPACETIC). To the best of our knowledge, our study is the first to show an association of the 15q24/25 locus with decline in lung function over time. Importantly, this genotype-associated difference in lung function decline was independent of baseline lung function level, indicating that variants in the nAChR
locus are not merely associated with an inherited lower lung function level, but also with an accelerated decline in lung function. Remarkably, no such effect was observed in former smokers. Based on power calculations, which revealed that our study had respectively 81.9% and 88.0% power for rs1051730 and rs8034191 to detect a significant difference in FEV1
/FVC decline in former smokers (Material S3), we believe that the absence of genetic variability in lung function decline in former smokers is a true negative finding. Importantly, we also established that the accelerated lung function decline in rs1051730 AA-carriers and rs8034191 GG-carriers may be relevant for clinical practice. For instance, compared to GG-carriers, homozygotes for the rs1051730 A-allele showed a two-fold increased risk (OR
2.29) to be diagnosed with very severe COPD GOLD IV and exhibited an OR of 5.0 to have end-stage COPD with need for lung transplantation 
. Likewise, GG-carriers of the rs8034191 genotype had a two-fold increased risk (OR
2.42) to have GOLD stage IV disease and a four-fold increased risk (OR
4.06) to be in need of lung transplantation compared to AA-carriers. Overall, these data suggest that current smokers carrying two copies of the at-risk alleles in the nAChR genes have an accelerated lung function decline, which may reflect susceptibility towards the development of severe COPD. The association between the same at-risk genotypes and very severe COPD supports this hypothesis. Future experiments are now warranted to investigate the association between genetic variation in the nAChR genes, lung function decline and incident COPD.
The observed associations may be biased for differences in smoking behavior between the nAChR
at-risk and wild type genotypes. Indeed, recent GWA studies have indicated that the nAChR
risk variants increase the risk of smoking addiction, presumably by mediating addictive effects in the brain, and promote more intense smoking, as reflected by a higher level of tobacco-specific nitrosamines per cigarette smoked in homozygous carriers of the at-risk alleles 
. However, we did not find an association between nAChR
variants and nicotine addiction related variables in our study population. Furthermore, the observed association between nAChR
variants and lung function decline withstood correction for smoking status (current versus former smokers), years-quit smoking and pack-years smoked. Nicotinic acetylcholine receptors (nAChR
s), which are encoded by the CHRNA
genes, are also widely expressed on airway epithelial cells and immune cells, such as macrophages, and their role in mediating inflammatory processes has been established 
. It is therefore possible that nAChR
s directly affect lung parenchyma and that genetic variation modulates the inflammatory response upon stimulation of nAChR
s by its agonists, thereby determining the extent of lung function decline. To completely discriminate between a direct and indirect effect, the relationship between the 15q24/25 locus and COPD could be studied among never-smokers. However, this approach is difficult given the very small number of never-smoking COPD patients. Alternatively, new statistical approaches could be applied, such as for instance mediation analysis. By use of this technique, Wang et al. established a direct association between rs1051730 and COPD risk (P
0.046), but also an indirect effect mediated by the variability in smoking behavior according to rs1051730 genotypes (0.006) 
. These findings are similar to our results: we did not establish an association between the 15q24/25 locus and nicotine addiction related variables. However, we demonstrated that rs1051730 affects lung function decline only in the group of active smokers, suggesting that this SNP exerts a mediating, but not a causal influence of smoking behavior. The hypothesis of a dual association (direct and indirect via smoking behavior) between 15q24/25, lung function decline and COPD should be investigated in future studies.
Moreover, since our study was performed in subjects from Caucasian ancestry, we cannot make any assumptions on the association between nAChR variants and lung function parameters or COPD in other ethnic groups. In a recent study in a Chinese Han population, no significant association was found between rs1051730 or rs8034191 and COPD in either former or current smokers. On the other hand, rs8034191 and rs1051730 were both associated with FEV1% predicted and FEV1/FVC in COPD cases 
The biology, by which rs1051730 and rs8034191 contribute to smoking-related disease phenotypes still remains unresolved. The rs1051730 SNP is a synonymous SNP located in exon 5 of the CHRNA3
gene, which is in strong linkage disequilibrium with a non-synonymous variant rs16969968 in exon 5 of the CHRNA5
and with rs55853698 in the promoter region of CHRNA 5 (all pairwise r2
>0,96; ) 
. The SNP rs16969968 results in an amino acid change (D398N) in the alpha5 receptor subunit protein and has been shown to affect receptor function 
. The location of the rs55853698 variant makes it a candidate for affecting mRNA transcription. On the other hand, rs1051730 and rs8034191 are also strongly linked with rs2568494 in the IREB2 gene (r2
0.692 and r2
0.790, respectively; ). Therefore genetic variation in the 15q24/25 region can also result in an altered function of the IREB2 gene. The finding of increased IREB2 protein and mRNA in lung-tissue samples from COPD subjects in comparison to controls supports a role of the IREB2 gene in COPD pathogenesis 
. Additional functional analyses are therefore required to establish whether the CHRNA3, CHRNA5 or IREB2 genes are involved in COPD development. Moreover, not only genetic variation, but also the impact of epigenetic variation in these genes on the development of COPD should be investigated. For instance in lung cancer, it has been shown that the CHRNA3 gene is frequently hypermethylated in lung tumor tissue samples (in comparison with blood control samples) and that this hypermethylation, by inducing gene silencing, results in resistance against nicotine-induced apoptosis 
Linkage Disequilibrium Map for the COPD-associated variants in the 15q24/25 region.
Regardless of the mechanisms by which nAChR
variants predispose to an accelerated lung function decline in smokers, the identification of this genetic locus could have important clinical implications. First of all, this marker would contribute to the prospective identification of a subset of susceptible smokers at high risk for an accelerated loss of lung function. Secondly, since smoking cessation is the most effective way to reduce the rate of lung function decline, this information could be used to convince subjects to quit smoking before symptoms of COPD develop. Remarkably, we did not identify a significant association between genetic variation in the nAChR
genes and FEV1
decline. However, as also suggested in another genetic association study on COPD-related phenotypes, it is likely that different genetic loci control FEV1
/FVC, since both parameters reflect a different functional measure 
The major strength of the present study is that we were able to access a large number of apparently healthy, but heavy smokers, as well as a relatively large cohort of symptomatic COPD patients, including a significant number of patients with end-stage lung disease in need of lung transplantation. This enabled us to study the role of nAChR genetic variants in various stages of COPD severity. Furthermore, the large number of study participants allowed extensive corrections for potentially confounding factors such as pack-years and smoking status. Unfortunately, we were not able to correct for smoking intensity (for instance cigarettes smoked per day). Some other limitations need to be acknowledged as well. First, COPACETIC only recruited heavy smokers and we could therefore not assess the effects of the 15q24/25 locus in subjects being exposed to less nicotine. Second, our follow-up period was limited to three years and we only assessed lung function level at two different time points. Third, in the COPACETIC cohort only males were included. Future studies should include females to assess the interaction between rs1057130/rs8034191, smoking status and lung function decline since it is known that differences in smoking behavior and resulting lung function disturbances exist between the sexes. Lastly, we only had information on the smoking status at baseline. In theory some quitters could have started smoking again which could have influenced our results.
In conclusion, we have demonstrated that in a European population heavy smokers carrying homozygous at-risk alleles for rs1051730 and rs8034191 in the nAChR locus are characterized by an accelerated decline in lung function, possibly leading to an increased risk of developing severe COPD. We thus provide one of the first genetic markers predictive for lung function decline.