American Journal of Respiratory and Critical Care Medicine

Rationale: Genome-wide association studies have identified genetic variants in the nicotinic acetylcholine receptor (nAChR) on chromosome 15q24/25 as a risk for nicotine dependence, lung cancer, and chronic obstructive pulmonary disease (COPD). Assessment of bronchial obstruction by spirometry, typically used for diagnosing COPD, fails, however, to detect emphysema.

Objectives: To determine the association of the 15q24/25 locus with emphysema.

Methods: The rs1051730 variant on 15q24/25 was genotyped in two independent white cohorts of 661 and 456 heavy smokers. Participants underwent pulmonary function tests and computed tomography (CT) of the chest, and took questionnaires assessing smoking behavior and health status.

Measurements and Main Results: The rs1051730 A-allele correlated with reduced FEV1 and with increased susceptibility for bronchial obstruction with a pooled odds ratio (OR) of 1.33 (95% confidence interval [CI] = 1.11–1.61; P = 0.0026). In both studies a correlation between the rs1051730 A-allele and lung diffusing capacity (DlCO) and diffusing capacity per unit alveolar volume (Kco) was observed. Consistently, the rs1051730 A-allele conferred increased risk for emphysema as assessed by CT (P = 0.0097 and P = 0.019), with a pooled OR of 1.39 (CI = 1.15–1.68; P = 0.00051). Visual emphysema scores and scores based on densities quantified on CT were more pronounced in A-allele carriers, indicating that rs1051730 correlates with the severity of emphysema.

Conclusions: The 15q24/25 locus in nAChR is associated with the presence and severity of emphysema. This association was independent of pack-years smoking, suggesting that nAChR is causally involved in alveolar destruction as a potentially shared pathogenic mechanism in lung cancer and COPD.

Scientific Knowledge on the Subject

Genetic studies have identified common variants in the nicotinic acetylcholine receptor genes as risk factors for lung cancer, nicotine addiction, and chronic obstructive pulmonary disease. It is unknown whether these variants also confer an increased risk for emphysema.

What This Study Adds to the Field

We report that a nicotinic acetylcholine receptor gene variant is associated with spirometric bronchial obstruction and emphysema assessed by computed tomography, independently from pack-years. This data supports a role for this receptor in smoke-induced alveolar destruction.

Three genome-wide association (GWA) studies recently identified common variants in the nicotinic acetylcholine receptor (nAChR) subunit genes on chromosome 15q24/25 as a risk factor for lung cancer (13). In one study, these variants also correlated with smoking quantities, nicotine dependence, and peripheral artery disease, for which smoking is the greatest modifiable risk factor (2). Such association was not seen in two other studies (1, 3). It was therefore unclear whether the 15q24/25 locus associates with lung cancer indirectly through an effect on the central nervous system affecting nicotine addiction, or directly by inducing carcinogenesis locally in the lung tissue. Subsequently, Young and colleagues reported that these lung cancer-associated variants are frequent in patients with bronchial obstruction (4), whereas Pillai and colleagues used a GWA approach to also identify 15q24/25 as a susceptibility locus for bronchial obstruction (5).

COPD classically involves two distinct spectra of clinical and pathological presentations: chronic bronchitis with bronchial obstruction, which involves airway inflammation with increased mucus production and airway remodeling, and emphysema, which is characterized by alveolar destruction (6, 7). The assessment of bronchial obstruction using spirometry, which is typically used for the diagnosis of COPD, fails to discriminate bronchial obstruction from emphysema—the latter being more reliably diagnosed using computed tomography (CT) (8, 9). As a result, most studies focusing on genetic susceptibility to COPD are restricted to airflow obstruction and fail to look at genetic predisposition to emphysema. Epidemiologic studies indicate, however, that bronchial obstruction and emphysema independently from each other predict the incidence of lung cancer (10). For instance, the risk for lung cancer is 1.8-fold increased in women with a prior history of bronchial obstruction, but as much as 6.4-fold when emphysema was diagnosed within 9 years before the lung cancer (11). Overall, this suggests that genetic studies in COPD should not only focus on disease diagnosed by spirometry, but should further distinguish between bronchial obstruction and emphysema phenotypes. Given the more explicit link between lung cancer and emphysema, susceptibility factors for lung cancer should also be tested in emphysema.

We therefore assessed association of the 15q24/25 locus with bronchial obstruction and emphysema in 661 heavy smokers from the LEUVEN cohort and replicated our findings in 456 heavy smokers from the COPACETIC cohort. Some of the methods and results of this study have been previously reported in the form of abstracts (12, 13).

Study Subjects

The LEUVEN cohort included 294 participants of the Dutch-Belgian randomized lung cancer screening trial (NELSON) who were recruited by the University Hospital of Leuven, Belgium (n = 294) (14). In addition, LEUVEN recruited 367 participants seen in the respiratory outpatient clinic, most of them with a former diagnosis of COPD (15). Inclusion criteria for the 661 participants were: a smoking history of 15 pack-years or more, older than 50 years of age, and the availability of a complete pulmonary function test, including lung diffusing capacity measurements. Exclusion criteria were: participants with suspicion or diagnosis of asthma, patients with other respiratory diseases, and patients with an exacerbation due to COPD within the last 6 weeks (n = 300). From all consenting participants, an extensive list of demographic variables, a complete pulmonary function assessment, CT scan of the chest, and questionnaires determining smoking history and quality of life (Clinical COPD Questionnaire [CCQ]) (16) were collected.

The COPACETIC study involved 456 participants from the NELSON trial recruited by the University Medical Centre in Utrecht, The Netherlands (14). In brief, participants were current and former smokers of 20 or more pack-years and aged between 50 and 75 years. Exclusion criteria were: a self-reported moderate or bad health status and a cancer diagnosed less than 5 years before recruitment. Patients with asthma were not excluded. For all 1,826 participants, a prebronchodilator spirometry, CT scan of the chest, and questionnaires assessing smoking history were available. Of these, 456 random participants also underwent lung diffusing capacity measurements and were selected for a GWA study. This subgroup was used in the present study.

The LEUVEN and COPACETIC studies were each approved by their local ethics committees and all participants provided written informed consent.

Pulmonary Function Testing

All pulmonary function measurements were performed with standardized equipment and according to American Thoracic Society and European Respiratory Society guidelines (17). Spirometric values in LEUVEN were postbronchodilator measurements, whereas in COPACETIC only prebronchodilator measurements were available. Diffusing capacity of the lung was determined by the single breath carbon monoxide gas transfer method (DlCO) and corrected for alveolar ventilation (Kco), but not for hemoglobin concentration (18). All variables are given as absolute values expressed as percent predicted of reference values (19, 20). Presence of bronchial obstruction was defined by postbronchodilator FEV1/FVC ratio less than 0.70 in the LEUVEN cohort and by prebronchodilator FEV1/FVC ratio less than 0.70 in the COPACETIC study, and severity of disease in both cohorts was staged by FEV1 expressed as percent predicted according to the latest Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification (21).

CT Imaging Protocol

The complete protocol used for CT imaging and quantification of emphysema in LEUVEN and COPACETIC is described in the data supplement (online data supplement note E1). Briefly, emphysema was semiquantitatively assessed by a visual scoring system in LEUVEN. Based on National Emphysema Treatment Trial criteria (22), four categories were generated yielding an alveolar destruction score ranging from 0 to 3 (no emphysema, emphysema affecting <20%, between 20–50%, and >50% of the lung, respectively). Thickening of the bronchial airway walls was scored on a semiquantitative three-level scale and presence or absence of bronchiectasis was assessed. In COPACETIC, all CT scans were analyzed using in-house developed software (23). The extent of emphysema was estimated using the percent of voxels with an apparent X-ray attenuation value below −910 HU and −950 HU. A cutoff value of greater than or equal to 1% of total lung volume attenuated below −950 HU was arbitrarily chosen as being abnormal and representing emphysema (24). A less stringent approach with a cutoff value of greater than or equal to 10% attenuated below −910 HU was used to define mild emphysema. The HU value per participant that delineates the 15% lowest lung density (15th percentile, abbreviated Perc15 HU) was also calculated.

Genotyping

Genotyping of the LEUVEN cohort for the rs1051730 variant in nAChR was performed in a blinded manner using iPLEX technology on a Sequenom MassARRAY (Sequenom Inc., San Diego, CA), as reported previously (25). Quality control was performed by genotyping 13 samples in duplicate with a duplicate concordance of 100%. For the replication analysis in COPACETIC, rs1051730 genotypes were obtained from Human610-Quad BeadChips (Illumina Inc., San Diego, CA). Additional information about the COPACETIC GWA study and the quality standards used are given in the online data supplement note E2.

Statistical Analysis

Data are summarized as frequencies (%) for categorical variables, and means (± SD) or medians (25th and 75th percentiles) for continuous variables. The proportion of lung voxels having less than −950 HU was ln-transformed before analysis. Differences in baseline characteristics between obstructive or emphysema participants and healthy smokers or between genotypes were compared by the Student t test, Mann-Whitney, or χ2 test. Differences in baseline characteristics between rs1051730 genotypes were compared by univariate analysis, Kruskal-Wallis, or χ2 test. Logistic regression was used to test the association of rs1051730 with presence of bronchial obstruction or emphysema, with or without adjustments for multivariables. Metaanalysis was performed with the Mantel-Haenszel and the DerSimonian and Laird method, and heterogeneity across the study populations was calculated using the χ2-based Cochran Q statistic (26). Statistical analyses were performed using SPSS software (v17.0). A two-sided P value less than 0.05 was considered statistically significant.

Population Characteristics

In total, 661 and 456 participants were included in the LEUVEN and COPACETIC cohorts. Genotyping for the rs1051730 variation succeeded in 659 (99.7%) participants from LEUVEN and 456 (100%) participants from COPACETIC. Observed genotypes were in Hardy-Weinberg equilibrium for both studies (P = 0.75 and P = 0.99, respectively).

Demographics, smoking history, pulmonary function tests, and CCQs are shown for all LEUVEN and COPACETIC participants and after stratification according to presence of bronchial obstruction (Table E1 and E2, respectively). In total, 552 (83.5%) participants from LEUVEN and 456 (100%) participants from COPACETIC also received a chest CT scan. Baseline variables stratified for the presence of emphysema based on CT scan are shown in Table 1. As expected, emphysema participants exhibited reduced pulmonary function in both cohorts. In LEUVEN, the participants exhibited more pronounced obstructive and emphysema phenotypes than COPACETIC participants. This most likely reflects the different inclusion criteria of both studies: COPACETIC mainly consists of asymptomatic smokers with (undiagnosed) early stages of bronchial obstruction, whereas LEUVEN additionally included participants with a clinical diagnosis of COPD. Smoking history did not differ between emphysema and healthy smoking participants, indicating that both groups were matched for smoking behavior.

TABLE 1. BASELINE CHARACTERISTICS OF THE LEUVEN AND COPACETIC COHORTS STRATIFIED FOR EMPHYSEMA



LEUVEN Cohort

COPACETIC Cohort

Total
Emphysema
No emphysema
P Value
Total
Emphysema
No emphysema
P Value
Subjects, no. (%)552310 (56.2%)242 (43.8%)456158 (34.6%)298 (65.3%)
Demographics
 Age, mean (SD), yr64.2 (7.9)65.0 (8.7)63.1 (6.5)0.00460.2 (5.5)61.1 (5.5)59.7 (5.5)0.006
 Male sex, no. (%)413 (74.9%)219 (70.9%)194 (80.2%)0.012431 (94.5%)152 (96.2%)279 (93.6%)0.25
 Height, mean (SD), cm169.9 (8.8)168.4 (9.0)171.8 (8.1)<0.001177.6 (7.3)178.1 (7.6)177.4 (7.1)0.30
Smoking
 Pack-years history, mean (SD), yr45.9 (23.4)47.3 (24.7)44.1 (21.8)0.1140.9 (18.4)41.1 (19.1)40.7 (16.9)0.83
 Current smokers, no. (%)225 (45.6%)109 (43.1%)116 (48.1%)0.26231 (50.7%)64 (40.5%)167 (56.0%)<0.001
 Smoked years, mean (SD), yr41.7 (9.0)42.7 (10.3)40.7 (7.6)0.01140.0 (8.5)38.6 (10.2)40.8 (7.3)0.014
 Pack-years/smoked years ratio, mean (SD)1.14 (0.51)1.19 (0.54)1.10 (0.51)0.0581.04 (0.50)1.07 (0.47)1.03 (0.52)0.073
 Years quit former smokers, median (25th–75th percentiles)7.0 (4.0–12.0)6.3 (3.0–12.5)8.0 (5.5–11.0)0.356.0 (4.0–10.0)7.0 (4.0–10.0)5.0 (3.0–10.0)0.11
Pulmonary function tests, mean (SD)
 FEV1, L2.03 (1.10)1.40 (0.85)2.78 (0.88)<0.0013.31 (0.74)3.20 (0.83)3.37 (0.68)0.026
 FEV1, % predicted69.9 (32.9)49.5 (26.3)92.3 (24.1)<0.00198.1 (18.2)94.7 (21.1)99.9 (16.3)0.008
 FVC, L3.47 (1.12)3.05 (1.06)3.94 (1.01)<0.0014.60 (0.86)4.79 (0.90)4.50 (0.81)<0.001
 FVC, % predicted94.5 (23.6)86.1 (23.5)104.2 (20.0)<0.001107.5 (15.4)117.7 (15.7)105.4 (14.9)<0.001
 FEV1/FVC ratio0.55 (0.18)0.44 (0.14)0.69 (0.11)<0.0010.72 (0.09)0.66 (0.10)0.75 (0.07)<0.001
 DlCO, mmol/min/kPa5.59 (2.47)4.15 (1.87)7.38 (1.89)<0.0018.50 (1.98)8.09 (2.11)8.71 (1.88)0.001
 DlCO, % predicted63.7 (24.8)48.4 (19.3)82.7 (16.4)<0.00187.5 (17.9)83.5 (20.2)89.6 (16.2)0.001
 Kco, mmol/min/kPa/L1.10 (0.32)0.92 (0.28)1.33 (0.21)<0.0011.25 (0.25)1.12 (0.24)1.32 (0.22)<0.001
 Kco, % predicted79.8 (24.1)66.3 (20.8)96.7 (15.8)<0.00182.7 (16.0)74.8 (15.7)86.9 (14.5)<0.001
Clinical COPD questionnaire, median (25th–75th percentiles)
 Symptom1.2 (0.5–2.2)2.0 (1.3–3.0)0.8 (0.3–1.5)<0.001N/AN/AN/AN/A
 Function state0.7 (0.0–2.5)2.1 (0.9–3.3)0.3 (0.0–0.8)<0.001N/AN/AN/AN/A
 Mental state0.0 (0.0–1.0)0.5 (0.0–1.5)0.0 (0.0–0.0)<0.001N/AN/AN/AN/A
 Total
2.2 (1.0–5.7)
5.3 (2.3–7.4)
1.0 (0.5–2.0)
<0.001
N/A
N/A
N/A
N/A

Definition of abbreviations: COPD = chronic obstructive pulmonary disease; DlCO = carbon monoxide diffusing capacity; Kco = carbon monoxide transfer coefficient; N/A = not applicable.

Of the 310 LEUVEN participants with emphysema, 50 (16.6%) participants were derived from the NELSON study and 212 (87.6%) participants were recruited from the respiratory outpatient clinic.

The rs1051730 A-Allele Correlates with Reduced Pulmonary Function

We first assessed whether the rs1051730 variant was associated with demographic parameters, smoking exposure, pulmonary function, and the clinical status from the study participants. Pack-years and current smoking status did not differ between rs1051730 genotypes in both cohorts, but significant differences were noticed at the pulmonary function level (Table 2).

TABLE 2. BASELINE CHARACTERISTICS ACCORDING TO RS1051730 GENOTYPES



LEUVEN Cohort

COPACETIC Cohort
rs1051730
GG
GA
AA
P Value
GG
GA
AA
P Value
N (%)259 (39.3%)301 (45.7%)99 (15.0%)200 (43.9%)203 (44.5%)53 (11.6%)
Demographics
 Age, years, mean (SD)64.8 (7.9)63.9 (7.7)63.2 (8.2)0.2859.8 (5.5)60.4 (5.6)60.8 (5.4)0.38
 Male sex, n (%)197 (76.1%)218 (72.7%)74 (74.7%)0.65189 (94.3%)191 (94.1%)50 (95.0%)0.957
 Height, mean (SD), cm169.9 (8.6)169.8 (9.0)169.5 (8.9)0.94179.0 (7.2)177.4 (7.4)177.3 (7.3)0.70
Smoking
 Pack-years history, mean (SD)44.5 (22.9)47.7 (24.2)44.1 (22.5)0.3139.1 (17.5)42.0 (19.2)44.0 (18.7)0.13
 Current smokers, n (%)106 (48.0%)120 (46.7%)34 (44.2%)0.84104 (52.0%)103 (50.7%)24 (45.3%)0.73
 Smoked years, mean (SD)42.1 (8.9)41.5 (8.8)40.9 (9.5)0.5339.9 (7.9)40.6 (8.5)38.1 (10.6)0.24
 Pack-years/smoked years ratio, mean (SD)1.11 (0.49)1.17 (0.52)1.17 (0.53)0.380.99 (0.46)1.07 (0.53)1.18 (0.49)0.055
 Years quit former smokers, median (25th−75th percentiles)8.0 (4.0–12.0)7.0 (4.0–11.0)8.0 (3.0–13.0)0.886.0 (4.0–10.0)6.0 (4.0–10.0)8.0 (4.0–11.0)0.82
Pulmonary function tests, mean (SD)
 FEV1, L1.99 (1.12)1.96 (1.11)1.74 (1.08)0.143.42 (0.74)3.25 (0.72)3.13 (0.79)0.012
 FEV1, % predicted67.8 (34.5)66.1 (33.3)59.1 (32.5)0.092100.4 (17.3)97.1 (18.9)93.0 (18.1)0.020
 FVC, L3.42 (1.15)3.37 (1.16)3.26 (1.10)0.514.64 (0.88)4.62 (0.83)4.73 (0.89)0.12
 FVC, % predicted92.8 (25.7)91.9 (24.0)89.4 (24.0)0.49107.6 (14.8)108.8 (16.1)102.8 (14.4)0.042
 FEV1/FVC ratio0.55 (0.18)0.55 (0.18)0.50 (0.19)0.0620.74 (0.08)0.70 (0.10)0.71 (0.09)0.0023
 DlCO, mmol/min/kPa5.67 (2.44)5.48 (2.51)4.82 (2.53)0.0188.87 (2.05)8.23 (1.88)8.15 (1.93)0.0024
 DlCO, % predicted64.5 (25.2)62.2 (24.8)54.9 (25.4)0.003890.8 (18.4)85.0 (16.8)84.7 (17.9)0.0019
 Kco, mmol/min/kPa/L1.12 (0.31)1.10 (0.47)0.99 (0.37)0.0301.31 (0.23)1.21 (0.25)1.22 (0.25)0.00026
 Kco, % predicted81.4 (23.1)78.3 (25.6)71.8 (27.5)0.006586.1 (15.2)79.9 (16.2)80.8 (16.1)0.00031
Clinical COPD questionnaire, median (25th–75th percentiles)
 Symptom1.3 (0.5–2.3)1.3 (0.5–2.3)1.6 (0.9–2.5)0.062N/AN/AN/AN/A
 Function state0.8 (0.0–2.0)0.8 (0.0–2.3)1.3 (0.5–3.0)0.084N/AN/AN/AN/A
 Mental state0.0 (0.0–0.5)0.0 (0.0–1.0)0.0 (0.0–1.0)0.35N/AN/AN/AN/A
 Total
2.3 (0.8–5.5)
2.3 (1.0–5.5)
3.5 (1.5–6.5)
0.092
N/A
N/A
N/A
N/A

Definition of abbreviations: COPD = chronic obstructive pulmonary disease; DlCO = carbon monoxide diffusing capacity; Kco = carbon monoxide transfer coefficient; N/A = not applicable.

The most consistent association was seen with lung diffusing capacity parameters. Mean DlCO and Kco values expressed in absolute values gradually decreased per A-allele (respectively, P = 0.018 and P = 0.030 for LEUVEN, and P = 0.0019 and P = 0.00026 for COPACETIC, Table 2). Adjustments for age, sex, height, pack-years, and years-quit revealed that this association was independent from known risk factors, both in LEUVEN (P = 0.004 and P = 0.016, respectively, for DlCO and Kco) and COPACETIC (P = 0.0031 and P = 0.00022). Similar associations were also observed for DlCO and Kco % predicted values (Table 2).

The rs1051730 genotypes were also correlated to the clinical COPD questionnaire in the LEUVEN participants. Although the overall effect on CCQ was not significant (P = 0.092; Table 2), rs1051730 AA carriers from LEUVEN exhibited a CCQ score that was 1.2 units higher than GG carriers (P = 0.035). This is an interesting observation because an increase of 0.4 units has previously been defined as the minimal clinically important difference (27). COPACETIC mainly consists of undiagnosed asymptomatic heavy smokers and therefore the CCQ score was not available for replication analysis

Association between rs1051730 and Bronchial Obstruction

We then assessed the association between rs1051730 and bronchial obstruction as defined by a FEV1/FVC ratio less than 0.70 (Table 3). In a crude analysis, the A-allele did not differ significantly between obstructive participants and healthy smokers from LEUVEN (P = 0.23), but was significantly more common in obstructive participants from COPACETIC (P = 0.0024). Meta-analysis of both studies under both fixed and random effect models (P for heterogeneity = 0.046) also resulted in an overall significant association between rs1051730 and bronchial obstruction (Table 3). However, the rs1051730 variant did not correlate with severity of COPD in LEUVEN or COPACETIC, because genotype distributions between the different GOLD stages were similar (Table E3). Overall, this demonstrates that rs1051730 was significantly associated with bronchial obstruction in the combined analysis as previously reported (4, 5), whereas no association with severity was noted.

TABLE 3. ASSOCIATION OF THE RS1051730 A-ALLELE WITH BRONCHIAL OBSTRUCTION AND EMPHYSEMA



Frequency of rs1051730-A Allele



Obstruction
No obstruction
OR (95% CI)
P Value
LEUVEN cohort369 (38.84%)130 (35.33%)1.17 (0.91–1.50)0.23
COPACETIC cohort127 (39.44%)182 (30.85%)1.55 (1.17–2.07)0.0024
Pooled (fixed effect)1.29 (1.07–1.56)0.00073
Pooled (random effect)1.30 (1.02–1.66)0.036
EmphysemaNo emphysema
LEUVEN cohort256 (41.29%)163 (33.68%)1.38 (1.08–1.77)0.0097
COPACETIC cohort123 (38.92%)186 (31.21%)1.40 (1.06–1.87)0.019
Pooled (fixed effect)1.39 (1.15–1.68)0.00051
Pooled (random effect)


1.39 (1.15–1.68)
0.00051

Definition of abbreviations: CI = confidence interval; OR = odds ratio.

Association of rs1051730 with Emphysema

Because rs1051730 correlated with lung diffusing capacities, we further tested the association of rs1051730 with emphysema as diagnosed by CT. In the LEUVEN cohort, we observed that the rs1051730 A-allele was significantly more common in participants with emphysema compared with those without (P = 0.0097; Table 3). At the genotypic level, rs1051730 increased risk according to an additive risk effect, with AA and GA genotypes being increasingly more common than GG carriers (69.4% and 55.1% vs. 51.8%, respectively; P = 0.021; Table 4). The association of rs1051730 with emphysema was also independent of other risk factors, because a multivariable-adjusted regression analysis correcting for age, sex, height, smoked pack-years, and years-quit also revealed a significant effect (OR = 1.40; P = 0.016).

TABLE 4. CHEST COMPUTED TOMOGRAPHY SCAN FINDINGS ACCORDING TO RS1051730 GENOTYPES




rs1051730


Total
GG
GA
AA
P Value
LEUVEN cohort552 (100%)218 (39.5%)249 (45.1%)85 (15.4%)
 Emphysema, n (%)310 (56.2%)113 (51.8%)138 (55.4%)59 (69.4%)0.021
 No emphysema, n (%)242 (43.8%)105 (48.2%)111 (44.6%)26 (30.6%)
 Visual emphysema score, median (25th–75th percentiles)
  RUL, %5 (0–50)3 (0–50)0 (0–50)20 (0–75)0.016
  RML, %0 (0–40)0 (0–40)0 (0–40)10 (0–50)0.0094
  RLL, %0 (0–40)0 (0–35)0 (0–35)5 (0–45)0.034
  LUL, %5 (0–45)0 (0–40)0 (0–40)18 (0–70)0.0026
  LML, %0 (0–40)0 (0–40)0 (0–30)10 (0–60)0.0049
  LLL, %0 (0–30)0 (0–30)0 (0–25)5 (0–35)0.072
  Tissue score, %2.5 (0.0–43.3)0.8 (0.0–40.8)2.5 (0.0–39.2)15.8 (0.0–55.0)0.015
 Alveolar destruction, n (%)*0.055
  0242 (43.8%)105 (43.4%)111 (45.9%)26 (10.7%)
  1110 (19.9%)35 (31.8%)56 (50.9%)19 (17.3%)
  292 (16.7%)40 (43.5%)36 (39.1%)16 (17.4%)
  3108 (19.6%)38 (35.2%)46 (42.6%)24 (22.2%)
 Bronchial thickening, n(%)288 (52.2%)117 (53.7%)124 (49.6%)48 (55.2%)0.56
 Bronchiectasis, n (%)113 (20.5%)48 (22.0%)46 (18.4%)20 (23.0%)0.52
COPACETIC cohort456 (100%)200 (43.9%)203 (44.5%)53 (11.6%)
 Proportion of voxels <−950 HU (25th−75th percentiles)0.59 (0.22–1.41)0.51 (0.18–1.13)0.67 (0.26–1.79)0.62 (0.21–1.99)0.00058
 Emphysema, n (%)158 (34.6%)56 (28.0%)81 (39.9%)21 (39.6%)0.031
 No emphysema, n (%)298 (65.3%)144 (72.0%)122 (60.1%)32 (60.4%)
 Mild emphysema, n (%)262 (57.4%)102 (51.0%)131 (64.5%)29 (54.7%)0.021
 No mild emphysema, n (%)194 (42.5%)98 (49.0%)72 (35.5%)24 (45.3%)
 Perc15 HU, (SD)
−905.6 (25.0)
−901.4 (24.3)
−909.4 (25.5)
−907.0 (23.2)
0.0056

Definition of abbreviations: LLF = left lower field; LMF = left middle field; LUF = left upper field; RLF = right lower field; RMF = right middle field; RUF = right upper field.

Perc15 HU denotes the HU value per participant that delineates the 15% lowest lung density. Alveolar destruction is a categorical classification of the tissue score.

*Percentages are row percentages; all other percentages are column percentages.

This association with emphysema was then replicated in COPACETIC. Because CT scans were quantitatively assessed in COPACETIC, we first correlated rs1051730 to proportion of lung voxels having less than −950 HU. Both uncorrected and corrected (for age, sex, height, pack-years, and years-quit) analyses revealed that rs1051730 correlated significantly with the proportion of lung voxels below −950 HU (P = 0.00058 and P = 0.0028, respectively). When subsequently defining emphysema as having greater than or equal to 1% of the total lung volume attenuated below −950 HU, the at-risk A-allele was significantly more common in participants with emphysema compared with those without (38.9 vs. 31.2%; P = 0.019). At the genotypic level, 39.6 and 39.9% of the AA and GA genotypes developed emphysema, compared with only 28.0% of the GG genotypes (P = 0.031; Table 4). In accordance with the LEUVEN data, association of rs1051730 with emphysema in COPACETIC was independent of other risk factors for emphysema, as demonstrated by multivariable-adjusted regression analysis (OR = 1.65; P = 0.018). Similar findings were observed using a less stringent radiographic cutoff (i.e., ≥10% of lung volume attenuated below −910 HU) to define emphysema (P = 0.021; Table 4).

Finally, meta-analysis of both the LEUVEN and COPACETIC studies under fixed- and random-effect models revealed a highly significant association between the rs1051730 A-risk allele and emphysema, with a pooled OR for both models of 1.39 (P = 0.00051; P for heterogeneity = 0.91; Table 3).

Association of rs1051730 with Severity of Emphysema

Stratification of visual emphysema scores into the six lung fields analyzed in LEUVEN revealed that AA carriers exhibited increased emphysema scores in almost each of the fields (Table 4). Remarkably, the tissue score, which averages the emphysema scores of each individual field, also increased gradually from GG carriers over GA to AA carriers (0.8, 2.5, and 15.8%, respectively; P = 0.015; Table 4). Moreover, classification of the tissue score into four categories of increasing severity revealed that AA genotypes were twice as common in participants with severe alveolar destruction versus no apparent destruction (22.4 vs. 10.7%; P = 0.016; Table 4). On the other hand, rs1051730 was not associated with bronchial thickening (bronchiectasis), indicating lack of an association with inflammation-mediated bronchial pathology.

To obtain a measure that reflects severity of emphysema within COPACETIC, the 15th percentile point (or Perc15 HU), a quantitative and continuous trait of emphysema defined as the cutoff value in HUs below which 15% of all voxels is distributed, was calculated for each participant, with a lower value representing a more damaged lung parenchyma. The AA and GA carriers exhibited a significantly lower Perc15 HU level compared with GG carriers (−907 HU and −909 HU vs. −901 HU, respectively; P = 0.0056; Table 4). In addition, univariate analysis corrected for age, height, smoked pack-years, and years-quit confirmed that association between rs1051730 and Perc15 HU was independent of other emphysema-related risk factors (P = 0.0027). Overall, this indicates that rs1051730 correlates with the severity of emphysema in both LEUVEN and COPACETIC.

Stratified Analysis of Isolated Bronchial Obstruction and Emphysema

To assess whether rs1051730 associates predominantly with bronchial obstruction, emphysema, or both, we performed a subgroup analysis and stratified LEUVEN and COPACETIC into four categories based on the presence or absence of bronchial obstruction and/or emphysema (Table 5). Allelic frequencies within each subgroup were compared with the control group, consisting of smokers free of bronchial obstruction and emphysema. In the combined analysis, we found that rs1051730 was most significantly associated with the presence of both bronchial obstruction and emphysema (OR = 1.51; P = 2.7 × 10−4; Table 5). Remarkably, we completely failed to see evidence for an association with isolated bronchial obstruction in the separate cohorts (OR = 1.03; P = 0.84; Table 5). Association with isolated emphysema was hard to assess, because this represented the smallest subgroup.

TABLE 5. OBSTRUCTION AND EMPHYSEMA PHENOTYPES ACCORDING TO RS1051730 ALLELES









Pooled
Leuven COPD cohort
COPACETIC Cohort
Fixed Effect
Random Effect
Phenotypes
Frequency of rs1051730 A-Allele
OR (95% CI)
P Value
Frequency of rs1051730 A-Allele
OR (95% CI)
P Value
OR (95% CI)
P Value
OR (95% CI)
P Value
Control98 (34.27%)Reference144 (30.6%)ReferenceReferenceReference
Obstruction65 (32.83%)0.94 (0.64–1.38)0.7442 (33.3%)1.13 (0.74–1.72)0.561.03 (0.78–1.36)0.841.03 (0.78–1.36)0.84
Obstruction and emphysema238 (40.75%)1.32 (0.98–1.77)0.06587 (43.9%)1.77 (1.26–2.50)9.6 × 10−41.51 (1.21–1.89)2.7 × 10−41.53 (1.08–2.18)0.018
Emphysema
18 (50.0%)
1.92 (0.95–3.85)
0.064
36 (30.5%)
0.99 (0.64–1.54)
0.98
1.26 (0.87–1.82)
0.22
1.38 (0.71–2.69)
0.34

Definition of abbreviations: CI = confidence interval; COPD = chronic obstructive pulmonary disease; CT = computed tomography; OR = odds ratio.

Cochran's P value testing heterogeneity for obstruction P = 0.49, obstruction and emphysema P = 0.047, and emphysema P = 0.028. The control group was defined as having no bronchial obstruction (FEV1/FVC ≥0.70) and no emphysema on CT, isolated obstruction as FEV1/FVC <0.70 with no emphysema on CT, isolated emphysema as no bronchial obstruction but emphysema on CT, and obstruction and emphysema as presence of both bronchial obstruction and emphysema on CT.

In the current study, we established that the at-risk A-allele of the rs1051730 variant on chromosome 15q24/25 confers an increased risk for bronchial obstruction (OR = 1.30) and emphysema (OR = 1.39). The rs1051730 variant also correlated with diffusing capacity measures DlCO and Kco, and the extent of alveolar destruction as visualized by CT. Notably, the risk effect for emphysema was independent from smoking behavior. We thus provide evidence for a hitherto unrecognized association between rs1051730, emphysema, and the severity thereof.

Emphysema is an important feature in the clinical spectrum of COPD and affects the majority of patients with COPD. Alveolar destruction due to emphysema also has important medical implications in COPD, because reduced oxygen uptake and loss of lung elasticity with concomitant airway collapsing and hyperinflation result in more dyspnea and a poor health-related quality of life (28). Although studies have shown that emphysema is always accompanied by small airway disease at the histopathological level (29), different phenotypes may exist at the clinical level. Indeed, in this study, most participants with emphysema also suffered from bronchial obstruction (65%), but a significant fraction of participants did not develop emphysema (21%) or developed emphysema but no obstruction (14%). This suggests that although both clinical phenotypes are likely to share common grounds, independent genetic factors contributing to either one of the phenotypes may exist (30). The question therefore arises whether rs1051730 is a risk factor for bronchial obstruction, emphysema, or both.

Based on the results of this study, rs1051730 associates with both emphysema and bronchial obstruction. In particular, the consistency of the association observed with emphysema was remarkable: rs1051730 correlated significantly with diffusing parameters DlCO and Kco both in LEUVEN and COPACETIC; rs1051730 also increased the risk for emphysema in both cohorts and correlated with the extent of alveolar destruction. Notably, emphysema and the severity thereof were assessed by two independent methods (i.e., semiquantitatively by a visual scoring system in LEUVEN and quantitatively using computer-assisted quantification in COPACETIC). The fact that the association of rs1051730 with emphysema was demonstrated in two independent cohorts implicates that it is robust. Together with hereditary α1-antitrypsin deficiency, this makes rs1051730 one of the few genetic risk factors so far established for emphysema (31, 32).

In COPACETIC, rs1051730 also associated with bronchial obstruction, but in LEUVEN this association was less pronounced. However, because rs1051730 has also been identified in a recent GWA study for COPD characterized by airway obstruction (5) and has been correlated with FEV1 in the 1958 British Birth Cohort (5), rs1051730 variant can indeed be considered as a susceptibility factor for bronchial obstruction.

Importantly, both associations were observed when bronchial obstruction and emphysema were considered as separate, independent phenotypes. However, when stratifying both cohorts into participants with isolated obstruction, isolated emphysema, or the combination of both, association of rs1051730 was more pronounced in participants exhibiting both obstruction and emphysema. Importantly, no evidence for an association with isolated bronchial obstruction was observed in COPACETIC or LEUVEN (OR = 0.91). On a more general level, this also suggests that future genetic studies in COPD should take clinical heterogeneity of the disease phenotype into account, in particular by differentiating between bronchial obstruction and emphysema.

The biology by which the rs1051730 variant contributes to these smoking-related disease phenotypes still remains unresolved. The rs1051730 single nucleotide polymorphism is located in exon 5 of the CHRNA3 gene, which is in strong linkage disequilibrium (r2 = 0.90 in the HapMap project, up to 0.94 in other studies) with a nonsynonymous variant rs16969968 in exon 5 of the CHRNA5 gene (33). Nicotinic acetylcholine receptor subunit genes (nAChRs) encode for receptors expressed in neurons of the mesolimbic pathway, but also in other tissues, such as bronchial and alveolar epithelial cells, pulmonary neuroendocrine cells, and lung cancer cell lines (22, 34). Recently, a correlation between rs1051730 and the level of the metabolite of nicotine, cotinine, was found, indicating that rs1051730 regulates nicotine intake among smokers (35). Intriguingly, it has also been proposed that nAChR binds N′-nitrosonornicotine and potential lung carcinogens such as nitrosamines (36, 37). Together with the fact that nAChR is expressed in lung cancer cells as a regulator of proliferation and apoptosis (38), our results suggest that rs1051730 also directly triggers alveolar destruction. Moreover, antagonists of the α7-subunit of nAChR were recently shown to have antitumoral effects in a mouse model of non-small cell lung cancer, thereby suggesting that nAChR indeed exerts direct effects (39). Obviously it is of paramount importance to determine how nAChR acts as disease susceptibility factor. If genetic variability in nAChR only determines nicotine intake and smoking addiction, the rs1051730 variant may independently increase susceptibility of all smoking-related diseases, including emphysema and COPD (2). However, if rs1051730 also directly affects the lung parenchyma, differential risk effects between these phenotypes may exist and rs1051730 could preferentially associate with a concurrent phenotype of both bronchial obstruction and emphysema, as observed in the current study.

We failed to see a clear association between rs1051730 and nicotine addiction, as measured by the number of pack-years smoked. This further supports the hypothesis that rs1051730 in nAChR directly affects the lung parenchyma. However, pack-years only partially capture smoking behavior and many other factors, including depth of inhalation, number of puffs per cigarette, and age at which smoking was started, also affect total nicotine exposure. It can therefore be argued that pack-years lack adequate precision and that for instance the Fagerstrom Test for Nicotine Dependence might be a better tool. The use of more sensitive measures is also necessary to exclude that small residual differences in smoking effects may have produced the observed differences in DlCO. Another explanation could be that only participants with a smoking history of 15 pack-years or more were included in LEUVEN and COPACETIC, thereby narrowing the spectrum of the nicotine dependence trait and reducing the ability to find an association. This most likely also explains why pack-years were not a risk factor for emphysema in this study.

Strengths of the current study are the extensive characterization of pulmonary function, the availability of CT scans in two independent cohorts, and the use of complementary assessments of emphysema (i.e., lung diffusion measurement and emphysema based on CT scan). However, some limitations should also be acknowledged. First, CT scans were scored differently between both cohorts: in LEUVEN a visual scoring system on different types of CT scans was used, whereas in COPACETIC a quantitative scoring method on a single CT acquisition protocol was applied. The latter is preferable, because it reduces interpretation bias to its minimum. On the other hand, association of rs1051730 with emphysema scored by both methods can also be regarded as an advantage, because it provides an independent validation of both methods used. Another limitation relates to the clinical relevance of rs1051730. Compared with carriers of the GG genotype, Kco values decreased by 11.6% in AA carriers from LEUVEN and by 6.8% in AA carriers from COPACETIC. Because no studies in COPD to our knowledge have carefully addressed which “minimal” decrease in diffusing capacity can be considered as clinically relevant, it remains unclear to which extent these reductions are clinically important and also may affect the patient's quality of life. Finally, it should be noted that LEUVEN, due to the additional inclusion of patients with a clinical diagnosis of COPD, did not exhibit a population-based study design. As a result, risk estimates between the marker (rs1051730) and disease (emphysema) may be biased, especially when the marker and the secondary trait (nicotine addiction) are both known to influence disease risk (40). However, because most associations have been corrected for emphysema-related risk factors, including the number of pack-years, we should have accounted for this possible bias.

Overall, the conclusion of the current study is that rs1051730 on chromosome 15q24/25 is associated with the presence and severity of emphysema. Furthermore, this association was independent from smoking exposure, suggesting that rs1051730 directly affects alveolar destruction in the lung.

The authors thank Kristien Debent, Claudia Carremans, Geert Celis, Bart Claes, Gilian Peuteman, Joanna Smolonska, Matthieu Platteel, Marike Boezen, Bart-Jan de Hoop, Bram van Ginneken, and Jan-Willem Lammers for their excellent assistance in the data collection, blood sampling, DNA extraction, and genotyping. The authors also thank the lung function and radiology technicians from the UZ Leuven and UMCU for their dedication and efforts.

1. Hung RJ, McKay JD, Gaborieau V, Boffetta P, Hashibe M, Zaridze D, Mukeria A, Szeszenia-Dabrowska N, Lissowska J, Rudnai P, et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature 2008;452:633–637.
2. Thorgeirsson TE, Geller F, Sulem P, Rafnar T, Wiste A, Magnusson KP, Manolescu A, Thorleifsson G, Stefansson H, Ingason A, et al. A variant associated with nicotine dependence, lung cancer and peripheral arterial disease. Nature 2008;452:638–642.
3. Amos CI, Wu X, Broderick P, Gorlov IP, Gu J, Eisen T, Dong Q, Zhang Q, Gu X, Vijayakrishnan J, et al. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat Genet 2008;40:616–622.
4. Young RP, Hopkins RJ, Hay BA, Epton MJ, Black PN, Gamble GD. Lung cancer gene associated with COPD: triple whammy or possible confounding effect? Eur Respir J 2008;32:1158–1164.
5. Pillai SG, Ge D, Zhu G, Kong X, Shianna KV, Need AC, Feng S, Hersh CP, Bakke P, Gulsvik A, et al. A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet 2009;5:e1000421.
6. Yoshida T, Tuder RM. Pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease. Physiol Rev 2007;87:1047–1082.
7. Molfino NA. Current thinking on genetics of chronic obstructive pulmonary disease. Curr Opin Pulm Med 2007;13:107–113.
8. Baraldo S, Saetta M, Cosio MG. Pathophysiology of the small airways. Semin Respir Crit Care Med 2003;24:465–472.
9. Buist AS, McBurnie MA, Vollmer WM, Gillespie S, Burney P, Mannino DM, Menezes AM, Sullivan SD, Lee TA, Weiss KB, et al. International variation in the prevalence of COPD (the BOLD study): a population-based prevalence study. Lancet 2007;370:741–750.
10. Wilson DO, Weissfeld JL, Balkan A, Schragin JG, Fuhrman CR, Fisher SN, Wilson J, Leader JK, Siegfried JM, Shapiro SD, et al. Association of radiographic emphysema and airflow obstruction with lung cancer. Am J Respir Crit Care Med 2008;178:738–744.
11. Schwartz AG, Cote ML, Wenzlaff AS, Van Dyke A, Chen W, Ruckdeschel JC, Gadgeel S, Soubani AO. Chronic obstructive lung diseases and risk of non-small cell lung cancer in women. J Thorac Oncol 2009;4:291–299.
12. Zanen P, Lammers JW, Postma D, Wijmenga C, Groen H, Boezen M, Vestbo J, Nordestgaard B, Nizankowska-Mogilnicka E, Eichinger M, et al. COPACETIC: unraveling the genetics of COPD. Presented at the European Respiratory Society Annual Congress, October 4–8 2008, Berlin, Abstract P522.
13. Smolonska J, Boezen HM, Groen H, Dijkstra AE, Postma DS, Oudkerk M, de Hoop B, van Ginneken B, Mali W, Lammers JW, et al. COPACETIC consortium. COPACETIC, a genome-wide association study on chronic obstructive pulmonary disease (COPD). Presented at the 59th Annual Meeting of The American Society of Human Genetics, October 24, 2009, Honolulu, HI, Abstract P845.
14. van Iersel CA, de Koning HJ, Draisma G, Mali WP, Scholten ET, Nackaerts K, Prokop M, Habbema JD, Oudkerk M, van Klaveren RJ. Risk-based selection from the general population in a screening trial: selection criteria, recruitment and power for the Dutch-Belgian randomised lung cancer multi-slice CT screening trial (nelson). Int J Cancer 2007;120:868–874.
15. Janssens W, Bouillon R, Claes B, Carremans C, Lehouck A, Buysschaert I, Coolen J, Mathieu C, Decramer M, Lambrechts D. Vitamin D deficiency is highly prevalent in COPD and correlates with variants in the vitamin d binding gene. Thorax 2009; (In Press).
16. van der Molen T, Willemse BW, Schokker S, ten Hacken NH, Postma DS, Juniper EF. Development, validity and responsiveness of the clinical COPD questionnaire. Health Qual Life Outcomes 2003;1:13.
17. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R, Enright P, van der Grinten CP, Gustafsson P, et al. Standardisation of spirometry. Eur Respir J 2005;26:319–338.
18. Rosenberg E. The 1995 update of recommendations for a standard technique for measuring the single-breath carbon monoxide diffusing capacity (transfer factor). Am J Respir Crit Care Med 1996;154:265–266.
19. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows. Report working party standardization of lung function tests, European Community for Steel and Coal. Official statement of the European Respiratory Society. Eur Respir J Suppl 1993;16:5–40.
20. Cotes JE, Chinn DJ, Quanjer PH, Roca J, Yernault JC. Standardization of the measurement of transfer factor (diffusing capacity). Report working party standardization of lung function tests, European Community for Steel and Coal. Official statement of the European Respiratory Society. Eur Respir J Suppl 1993;16:41–52.
21. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007;176:532–555.
22. Wang Y, Pereira EF, Maus AD, Ostlie NS, Navaneetham D, Lei S, Albuquerque EX, Conti-Fine BM. Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors. Mol Pharmacol 2001;60:1201–1209.
23. Schilham AM, van Ginneken B, Gietema H, Prokop M. Local noise weighted filtering for emphysema scoring of low-dose ct images. IEEE Trans Med Imaging 2006;25:451–463.
24. Friedman PJ. Imaging studies in emphysema. Proc Am Thorac Soc 2008;5:494–500.
25. Buysschaert ID, Grulois V, Eloy P, Jorissen M, Rombaux P, Bertrand B, Collet S, Bobic S, Vlaminck S, Hellings PW, et al. Genetic evidence for a role of il33 in nasal polyposis. Allergy. (In press)
26. Lambrechts D, Poesen K, Fernandez-Santiago R, Al-Chalabi A, Del Bo R, Van Vught PW, Khan S, Marklund S, Brockington A, Van Marion I, et al. Meta-analysis of VEGF variations in ALS: increased susceptibility in male carriers of the -2578aa genotype. J Med Genet 2009;46:840–846.
27. Kocks JW, Tuinenga MG, Uil SM, van den Berg JW, Stahl E, van der Molen T. Health status measurement in COPD: the minimal clinically important difference of the clinical COPD questionnaire. Respir Res 2006;7:62.
28. Makita H, Nasuhara Y, Nagai K, Ito Y, Hasegawa M, Betsuyaku T, Onodera Y, Hizawa N, Nishimura M. Characterisation of phenotypes based on severity of emphysema in chronic obstructive pulmonary disease. Thorax 2007;62:932–937.
29. Hogg JC, Timens W. The pathology of chronic obstructive pulmonary disease. Annu Rev Pathol 2009;4:435–459.
30. Patel BD, Coxson HO, Pillai SG, Agusti AG, Calverley PM, Donner CF, Make BJ, Muller NL, Rennard SI, Vestbo J, et al. Airway wall thickening and emphysema show independent familial aggregation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;178:500–505.
31. Smolonska J, Wijmenga C, Postma DS, Boezen HM. Meta-analyses on suspected chronic obstructive pulmonary disease genes: a summary of 20 years' research. Am J Respir Crit Care Med 2009;180:618–631.
32. Wan ES, Silverman EK. Genetics of COPD and emphysema. Chest 2009;136:859–866.
33. Saccone SF, Hinrichs AL, Saccone NL, Chase GA, Konvicka K, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau O, et al. Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. Hum Mol Genet 2007;16:36–49.
34. Minna JD. Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer. J Clin Invest 2003;111:31–33.
35. Keskitalo K, Broms U, Heliovaara M, Ripatti S, Surakka I, Perola M, Pitkaniemi J, Peltonen L, Aromaa A, Kaprio J. Association of serum cotinine level with a cluster of three nicotinic acetylcholine receptor genes (chrna3/chrna5/chrnb4) on chromosome 15. Hum Mol Genet 2009;18:4007–4012.
36. Schuller HM. Nitrosamines as nicotinic receptor ligands. Life Sci 2007;80:2274–2280.
37. Schuller HM, Orloff M. Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells. Biochem Pharmacol 1998;55:1377–1384.
38. Schuller HM. Is cancer triggered by altered signalling of nicotinic acetylcholine receptors? Nat Rev Cancer 2009;9:195–205.
39. Paleari L, Negri E, Catassi A, Cilli M, Servent D, D'Angelillo R, Cesario A, Russo P, Fini M. Inhibition of nonneuronal alpha7-nicotinic receptor for lung cancer treatment. Am J Respir Crit Care Med 2009;179:1141–1150.
40. Monsees GM, Tamimi RM, Kraft P. Genome-wide association scans for secondary traits using case-control samples. Genet Epidemiol 2009;33:717–728.
Correspondence and requests for reprints should be addressed to Diether Lambrechts, M.Sc., Ph.D., Vesalius Research Center, K.U. Leuven, Campus Gasthuisberg, Herestraat 49 Box 912, B-3000, Leuven, Belgium. E-mail:

Related

No related items
American Journal of Respiratory and Critical Care Medicine
181
5

Click to see any corrections or updates and to confirm this is the authentic version of record