220 Original article
CHRNA3 and CYP3A5*3 genotype, lung function and chronic obstructive pulmonary disease in the general population Diljit Kaur-Knudsena,c, Stig E. Bojesena,b,c and Børge G. Nordestgaarda,b,c Objective Genetic variations are most likely an additional risk factor besides tobacco smoking per se for the risk of chronic obstructive pulmonary disease (COPD). In this study, we compared genetic variants influencing the effect of smoking on COPD, that is, the effect of the well-known splicing defect polymorphism, CYP3A5*3 (rs776746), identified before genome-wide association studies, with the genome-wide association studies identified CHRNA3 (rs1051730) polymorphism on the risk of decreased lung function and COPD. Materials and methods In all, 10 605 participants from the general population were genotyped. Information on spirometry, hospital admissions and smoking behaviour was recorded. Endpoints were lung function and COPD. Results For CHRNA3, the percentage of forced expiratory volume in 1 s (FEV1%) predicted was 89.3, 90.6 and 92.4% in homozygous, heterozygous and noncarrier ever-smokers (P-trend < 0.001). The corresponding values for forced vital capacity percentage (FVC%) predicted were 94.5, 95.2 and 96.7% (P-trend < 0.001), and for FEV1/FVC ratio, the values were 0.753, 0.760 and 0.764 (P-trend = 0.008). The odds ratio for COPD in homozygous versus noncarrier ever-smokers was 1.5 [95% confidence interval (CI) 1.3–1.9] for COPD hospitalization, 1.3 (95% CI 1.1–1.6) for COPD defined as FEV1/FVC less than
Introduction Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality in the general population. Although tobacco smoking is a well-recognized risk factor, not all smokers develop COPD. Some individuals seem to be more susceptible to the damaging chemicals in tobacco smoke, most likely because of genetic variation. Tobacco smoke contains polycyclic aromatic hydrocarbons (PAHs), which become activated in the body by the enzyme system cytochrome P450 [1]. The activated PAHs have been associated with decreased lung function [2]. In one of the PAH-metabolizing cytochrome P450 enzymes, CYP3A5 [3,4], a genetic variant CYP3A5*3, causes low expression of the variant allele [5–7]. The CYP3A5*3 (rs776746) is an exchange of A to G causing a splice defect in the transcript and is the common variant in Whites [5–7]. Hence, CYP3A5*3 could affect the activation of PAHs and Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pharmacogeneticsandgenomics.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 1744-6872
lower limit of normal, 1.3 (95% CI 1.0–1.5) for the Global Initiative for Chronic Obstructive Lung Disease category 1–4 (GOLD 1–4), 1.2 (95% CI 1.0–1.5) for GOLD 2–4 and 1.5 (95% CI 1.1–2.2) for GOLD 3–4. This association could not be found in never-smokers. No association was found for CYP3A5*3. Conclusion The CHRNA3 genotype is associated with decreased lung function and risk of COPD among ever-smokers, whereas this was not the case for CYP3A5*3. Pharmacogenetics and Genomics c 2014 Wolters Kluwer Health | Lippincott 24:220–229 Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:220–229 Keywords: CHRNA3, chronic obstructive pulmonary disease, CYP3A5, genetic, lung disease, polymorphism a Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, bCopenhagen City Heart Study, Frederiksberg Hospital, Copenhagen University Hospital and cInstitute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Herlev, Denmark
Correspondence to Børge G. Nordestgaard, MD, DMSc, Department of Clinical Biochemistry, 54M1, Herlev Hospital, Copenhagen University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark Tel: + 45 3868 3297; fax: + 45 3868 3311; e-mail: [email protected] Received 21 April 2013 Accepted 24 January 2014
thus lung function and risk of COPD in smokers, but this has not yet been examined. The CHRNA3–CHRNB4–CHRNA5 gene cluster, coding for a nicotinic acetylcholine receptor, has recently been linked to tobacco-related diseases such as lung cancer, bladder cancer, and COPD, with the most replicated polymorphism being the synonymous C to T exchange rs1051730 [8–13]. In this study, we compared the effect of the well-known splicing defect polymorphism CYP3A5*3 (rs776746), identified before genome-wide association studies (GWAS), with the GWAS identified CHRNA3 (rs1051730) polymorphism on the risk of decreased lung function and COPD among ever-smokers in the general population. To examine this, we genotyped 10 341 and 10 330 participants, respectively, from the Danish general population, the Copenhagen City Heart Study.
Materials and methods Setting and participants
All participants provided written informed consent and the studies were approved by Danish ethics committees DOI: 10.1097/FPC.0000000000000038
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
CHRNA3 and CYP3A5*3 genotypes and lung disease Kaur-Knudsen et al. 221
(KF-100 2039/91, KF-01-144/01). Studies were carried out according to the Declaration of Helsinki. The Copenhagen City Heart Study was initiated in 1976–1978. Participants older than 20 years of age were selected randomly from the general population of Copenhagen and reinvited to follow-up examinations in 1981–1983, 1991–1994 and 2001–2003. New and younger participants supplemented each follow-up examination, and at all four examinations, the participants filled in a self-administered questionnaire reviewed by an investigator on the day of attendance and underwent a physical examination. At the third (1991–1994) and fourth (2001–2004) examination, whole-blood samples were frozen and later DNA was isolated.
exposed to passive smoking on a daily basis. These three questions were combined as information on passive smoking in the Table of Characteristics. Spirometry and chronic obstructive pulmonary disease diagnoses
The TaqMan method (Applied Biosystems Inc., Foster City, California, USA) was used to genotype CYP3A5*3 (rs776746) and CHRNA3 (rs1051730). Primers and probes are available from the authors on request. Because of re-runs, genotyping call rates were higher than 99.8%.
Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were measured using a dry wedge spirometer (Vitalograph; Maids Moreton, Buckinghamshire, UK). Lung function was measured as prebronchodilator values. Predicted values were calculated using multiple regression analyses separately for men and women, with age and height as covariates in never-smokers; never-smoking individuals with asthma and/or COPD were not excluded before calculating these reference equations for lung function. The per cent predicted value was calculated by dividing the observed value with the predicted value. Lower limit of normal was calculated as the difference between the predicted value and 1.645 times the SE of the estimate separately for men and women. COPD was defined in five different ways: (a) hospitalization with COPD [International Classification of Diseases 8th ed. (ICD-8): 491–492; ICD-10: J41–J44], (b) below lower limit of normal for FEV1/FVC, (c) the Global Initiative for Chronic Obstructive Lung Disease category 1–4 (GOLD 1–4) being FEV1/FVC < 0.7, (d) GOLD 2–4 being FEV1/FVC < 0.7 and FEV1% < 80% predicted and (e) GOLD 3–4 being FEV1/FVC < 0.7 and FEV1% < 50% predicted. Individuals younger than 40 years of age with self-reported asthma were excluded from analyses of COPD.
Smoking behaviour
Statistical analyses
All participants were asked questions about smoking behaviour in a self-administered questionnaire, a questionnaire that was reviewed by the participant together with a study examiner on the day of attendance. On the basis of this information, the participants were divided into never-smokers or ever-smokers, ever-smokers being both current and former smokers. Ever-smokers were asked at what age they started smoking and if they were former smokers at what age they stopped smoking. They were also asked how many years they had smoked in total. Furthermore, ever-smokers were asked whether they had smoked cigarettes, cheroots, cigars and/or pipe tobacco and the number of grams smoked per day. One cigarette corresponds to 1 g of tobacco, a cheroot to 3 g, a cigar to 5 g and a pack of pipe tobacco to 50 g. Cumulative tobacco consumption was calculated for ever-smokers in pack-years, which was defined as smoking of 20 g of tobacco per day for a year. The participants were asked whether they had been exposed to dust and/or welding fumes at work. In the 1991–1994 examination, the participants were also asked whether any person in their household was a smoker or whether one of their parents used to be smokers. In the 2001–2003 examination, the participants were additionally asked whether they were
Data analyses were carried out using STATA/SE 11.1 (StataCorp LP, College Station, Texas, USA). We used the trend test and multiple regression. Odds ratios were calculated using logistic regression and adjusted for age and sex, or for age, sex and cumulative tobacco consumption; for the latter, missing data for cumulative tobacco consumption (n = 58) were imputed using age and sex. The P-values for interaction between genotype and cumulative tobacco consumption were calculated using a regression model where the simple model with lung function, genotype and cumulative tobacco consumption was nested in a model also containing a two-factor term (genotype cumulative tobacco consumption). Meta-analyses were carried out with our previous study [13] between the CHRNA3 genotype and lung function/COPD (Figs 4 and 5) using fixed and random-effect models. Mediation analysis was carried out using the CHRNA3 genotype as an independent variable, disease (COPD or lung function) as a dependent variable and cumulative tobacco consumption as a mediator variable. The indirect effect of genotype on lung function/COPD through cumulative tobacco consumption was determined using the product of the coefficients method.
In the present study, we included participants from the examinations in 1991–1994 and 2001–2003. Of 10 605 participants with available DNA, 261 (2.5%) participants were not of Danish descent and were therefore excluded from statistical analyses. For CYP3A5*3 and CHRNA3, the genotype was determined in 10 341 (5754 women and 4587 men) and 10 330 (5746 women and 4584 men) of a total 10 344 available participants with DNA. Genotyping
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
222 Pharmacogenetics and Genomics
2014, Vol 24 No 4
For CYP3A5*3, the most common allele among Whites is the G allele, and therefore, the CYP3A5*3 GG homozygous genotype group was used as the reference group when calculating risk estimates; however, the G allele is the allele with a splice defect and presumed lack of enzyme activity and therefore the AA genotype is called a noncarrier. For CHRNA3, the most common allele among Whites is the C allele, and therefore, the CHRNA3 CC homozygous genotype group was used as the reference group when calculating risk estimates.
Results We included a total of 10 330 participants with a known CHRNA3 genotype, of whom 44.6% were noncarriers, 44.3% were heterozygotes and 11.1% were homozygotes (Table 1). We included a total of 10 341 participants with a known CYP3A5*3 genotype, of whom 0.5% were noncarriers (homozygous for the rare functional allele in Whites), 13.1% were heterozygotes and 86.4% were homozygotes. These frequencies are comparable with those reported in previous studies of Whites [5] and both were in Hardy–Weinberg equilibrium (P = 0.79 for CHRNA3 and 0.37 for CYP3A5*3). Characteristics did not differ by genotype (Table 1). Information on detailed smoking behaviour and CHRNA3 genotype has been
Table 1
published previously, where an association was found with daily tobacco consumption (P = 0.0001), cumulative tobacco consumption (P = 0.003) and smoke inhalation (P = 0.001) [8]. Characteristics on detailed smoking behaviour for CYP3A5*3 did not differ by genotype (Supplementary Table 1; Supplemental digital content 1, http://links.lww.com/FPC/A705). Lung function
For CHRNA3, FEV1% predicted was 89.3, 90.6 and 92.4% in homozygous, heterozygous and noncarrier ever-smokers, respectively (P-trend < 0.001; Table 2). The corresponding values for FVC% predicted were 94.5, 95.2 and 96.7% (P-trend < 0.001), and for FEV1/FVC ratio, the values were 0.753, 0.760 and 0.764 (P-trend = 0.008), respectively. When adjusted for cumulative tobacco consumption, these associations remained significant. The results were very similar when adjusted for smoking status (current and former smokers instead of cumulative tobacco consumption; data not shown). No interaction was detected between genotype and cumulative tobacco consumption (all P > 0.05; Table 2). In never-smokers, FVC% predicted for CHRNA3 were 99.1, 99.6 and 100.5% in homozygous, heterozygous and noncarriers, respectively (P-trend = 0.03); however, this association disappeared when
Characteristics of participants by CHRNA3 and CYP3A5*3 genotypes in the Danish general population, the Copenhagen City Heart
Study
Characteristics
CHRNA3 (rs1051730)
CYP3A5*3 (rs776746)
Heterozygotes (CT)
Heterozygotes (AG)
Noncarriers (CC)
All participants [n (%)] 4611 (44.6) Women [n (%)] 2587 (56.1) Age (years) 59.0 (44.8–69.6) Ever-smokers [n (%)] 3471 (75.4) Exposure to dust and/or welding 860 (18.8) smoke at work [n (%)] Passive smoking [n (%)] 3633 (79.8)
4572 2562 59.5 3514 848
PHomozygotes (TT) trend Noncarriers (AA)
(44.3) 1147 (11.1) (56.0) 597 (52.0) (45.4–69.8) 59.2 (45.6–69.1) (77.0) 880 (76.8) (18.7) 249 (21.8)
3658 (80.8)
928 (82.1)
Homozygotes (GG)
58 (0.5) 1349 (13.1) 0.06 28 (48.3) 766 (56.8) 0.79 55.9 (37.3–68.6) 59.8 (45.6–69.3) 0.13 41 (70.7) 1014 (75.2) 0.11 11 (19.3) 250 (18.7)
8934 4960 59.2 6820 1697
0.06
7112 (80.7)
43 (74.1)
1064 (79.8)
(86.4) (55.5) (45.2–69.7) (76.5) (19.1)
Ptrend 0.71 0.52 0.17 0.75 0.26
Values are median (interquartile range) for age and number (%) for categorical variables. The P-values were calculated in a trend test.
Table 2 Lung function in ever-smokers by CHRNA3 and CYP3A5*3 genotypes in the Danish general population, the Copenhagen City Heart Study CHRNA3 (rs1051730)
FEV1% predicted FVC% predicted FEV1/FVC ratio
CYP3A5*3 (rs776746)
Noncarriers (CC) (N = 3412)
Heterozygotes (CT) (N = 3469)
Homozygotes (TT) (N = 869)
P-trend
P-value adjusted
P-value for interaction
Homozygotes (GG) (N = 6714)
Heterozygotes (AG) (N = 1005)
92.4±20.5
90.6±21.3
89.3±21.3
< 0.001
< 0.001
0.30
91.3±21.2
90.3±19.9
93.7±17.3
0.13
96.7±17.7
95.2±18.2
94.5±18.1
< 0.001
0.001
0.27
95.9±18.1
94.9±17.2
97.2±14.7
0.15
0.764±0.094
0.760±0.099
0.753±0.099
0.02
0.10
0.761±0.097
0.761±0.093
0.770±0.077
0.73
0.008
Noncarriers P(AA) (N = 41) trend
Data are mean±SD. P-trend was calculated with genotypes coded 0, 1 and 2. P-values adjusted were adjusted for cumulative tobacco consumption and calculated by multiple regression. P-values for interaction were between genotype and cumulative tobacco consumption. Because some individuals lacked information on spirometry, the numbers in this table and Table 3 do not add up to the total number of individuals genotyped, as mentioned in Table 1. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
CHRNA3 and CYP3A5*3 genotypes and lung disease Kaur-Knudsen et al. 223
adjusted for multiple comparison and thus no association was found between the CHRNA3 genotype and lung function in never-smokers (all P-trends > 0.05; Table 3). CYP3A5*3 did not associate with any lung function measurements in ever-smokers or never-smokers (all P-trends > 0.05; Tables 2 and 3).
Chronic obstructive pulmonary disease
The CHRNA3 genotype was associated with increased risk of COPD in ever-smokers from noncarriers to heterozygotes to homozygotes when adjusted for age and sex, irrespective of the definition used (P-trends = 0.02 to
CHRNA3 and CYP3A5*3 genotype, lung function and chronic obstructive pulmonary disease in the general population Diljit Kaur-Knudsena,c, Stig E. Bojesena,b,c and Børge G. Nordestgaarda,b,c Objective Genetic variations are most likely an additional risk factor besides tobacco smoking per se for the risk of chronic obstructive pulmonary disease (COPD). In this study, we compared genetic variants influencing the effect of smoking on COPD, that is, the effect of the well-known splicing defect polymorphism, CYP3A5*3 (rs776746), identified before genome-wide association studies, with the genome-wide association studies identified CHRNA3 (rs1051730) polymorphism on the risk of decreased lung function and COPD. Materials and methods In all, 10 605 participants from the general population were genotyped. Information on spirometry, hospital admissions and smoking behaviour was recorded. Endpoints were lung function and COPD. Results For CHRNA3, the percentage of forced expiratory volume in 1 s (FEV1%) predicted was 89.3, 90.6 and 92.4% in homozygous, heterozygous and noncarrier ever-smokers (P-trend < 0.001). The corresponding values for forced vital capacity percentage (FVC%) predicted were 94.5, 95.2 and 96.7% (P-trend < 0.001), and for FEV1/FVC ratio, the values were 0.753, 0.760 and 0.764 (P-trend = 0.008). The odds ratio for COPD in homozygous versus noncarrier ever-smokers was 1.5 [95% confidence interval (CI) 1.3–1.9] for COPD hospitalization, 1.3 (95% CI 1.1–1.6) for COPD defined as FEV1/FVC less than
Introduction Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality in the general population. Although tobacco smoking is a well-recognized risk factor, not all smokers develop COPD. Some individuals seem to be more susceptible to the damaging chemicals in tobacco smoke, most likely because of genetic variation. Tobacco smoke contains polycyclic aromatic hydrocarbons (PAHs), which become activated in the body by the enzyme system cytochrome P450 [1]. The activated PAHs have been associated with decreased lung function [2]. In one of the PAH-metabolizing cytochrome P450 enzymes, CYP3A5 [3,4], a genetic variant CYP3A5*3, causes low expression of the variant allele [5–7]. The CYP3A5*3 (rs776746) is an exchange of A to G causing a splice defect in the transcript and is the common variant in Whites [5–7]. Hence, CYP3A5*3 could affect the activation of PAHs and Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pharmacogeneticsandgenomics.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 1744-6872
lower limit of normal, 1.3 (95% CI 1.0–1.5) for the Global Initiative for Chronic Obstructive Lung Disease category 1–4 (GOLD 1–4), 1.2 (95% CI 1.0–1.5) for GOLD 2–4 and 1.5 (95% CI 1.1–2.2) for GOLD 3–4. This association could not be found in never-smokers. No association was found for CYP3A5*3. Conclusion The CHRNA3 genotype is associated with decreased lung function and risk of COPD among ever-smokers, whereas this was not the case for CYP3A5*3. Pharmacogenetics and Genomics c 2014 Wolters Kluwer Health | Lippincott 24:220–229 Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:220–229 Keywords: CHRNA3, chronic obstructive pulmonary disease, CYP3A5, genetic, lung disease, polymorphism a Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, bCopenhagen City Heart Study, Frederiksberg Hospital, Copenhagen University Hospital and cInstitute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Herlev, Denmark
Correspondence to Børge G. Nordestgaard, MD, DMSc, Department of Clinical Biochemistry, 54M1, Herlev Hospital, Copenhagen University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark Tel: + 45 3868 3297; fax: + 45 3868 3311; e-mail: [email protected] Received 21 April 2013 Accepted 24 January 2014
thus lung function and risk of COPD in smokers, but this has not yet been examined. The CHRNA3–CHRNB4–CHRNA5 gene cluster, coding for a nicotinic acetylcholine receptor, has recently been linked to tobacco-related diseases such as lung cancer, bladder cancer, and COPD, with the most replicated polymorphism being the synonymous C to T exchange rs1051730 [8–13]. In this study, we compared the effect of the well-known splicing defect polymorphism CYP3A5*3 (rs776746), identified before genome-wide association studies (GWAS), with the GWAS identified CHRNA3 (rs1051730) polymorphism on the risk of decreased lung function and COPD among ever-smokers in the general population. To examine this, we genotyped 10 341 and 10 330 participants, respectively, from the Danish general population, the Copenhagen City Heart Study.
Materials and methods Setting and participants
All participants provided written informed consent and the studies were approved by Danish ethics committees DOI: 10.1097/FPC.0000000000000038
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
CHRNA3 and CYP3A5*3 genotypes and lung disease Kaur-Knudsen et al. 221
(KF-100 2039/91, KF-01-144/01). Studies were carried out according to the Declaration of Helsinki. The Copenhagen City Heart Study was initiated in 1976–1978. Participants older than 20 years of age were selected randomly from the general population of Copenhagen and reinvited to follow-up examinations in 1981–1983, 1991–1994 and 2001–2003. New and younger participants supplemented each follow-up examination, and at all four examinations, the participants filled in a self-administered questionnaire reviewed by an investigator on the day of attendance and underwent a physical examination. At the third (1991–1994) and fourth (2001–2004) examination, whole-blood samples were frozen and later DNA was isolated.
exposed to passive smoking on a daily basis. These three questions were combined as information on passive smoking in the Table of Characteristics. Spirometry and chronic obstructive pulmonary disease diagnoses
The TaqMan method (Applied Biosystems Inc., Foster City, California, USA) was used to genotype CYP3A5*3 (rs776746) and CHRNA3 (rs1051730). Primers and probes are available from the authors on request. Because of re-runs, genotyping call rates were higher than 99.8%.
Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were measured using a dry wedge spirometer (Vitalograph; Maids Moreton, Buckinghamshire, UK). Lung function was measured as prebronchodilator values. Predicted values were calculated using multiple regression analyses separately for men and women, with age and height as covariates in never-smokers; never-smoking individuals with asthma and/or COPD were not excluded before calculating these reference equations for lung function. The per cent predicted value was calculated by dividing the observed value with the predicted value. Lower limit of normal was calculated as the difference between the predicted value and 1.645 times the SE of the estimate separately for men and women. COPD was defined in five different ways: (a) hospitalization with COPD [International Classification of Diseases 8th ed. (ICD-8): 491–492; ICD-10: J41–J44], (b) below lower limit of normal for FEV1/FVC, (c) the Global Initiative for Chronic Obstructive Lung Disease category 1–4 (GOLD 1–4) being FEV1/FVC < 0.7, (d) GOLD 2–4 being FEV1/FVC < 0.7 and FEV1% < 80% predicted and (e) GOLD 3–4 being FEV1/FVC < 0.7 and FEV1% < 50% predicted. Individuals younger than 40 years of age with self-reported asthma were excluded from analyses of COPD.
Smoking behaviour
Statistical analyses
All participants were asked questions about smoking behaviour in a self-administered questionnaire, a questionnaire that was reviewed by the participant together with a study examiner on the day of attendance. On the basis of this information, the participants were divided into never-smokers or ever-smokers, ever-smokers being both current and former smokers. Ever-smokers were asked at what age they started smoking and if they were former smokers at what age they stopped smoking. They were also asked how many years they had smoked in total. Furthermore, ever-smokers were asked whether they had smoked cigarettes, cheroots, cigars and/or pipe tobacco and the number of grams smoked per day. One cigarette corresponds to 1 g of tobacco, a cheroot to 3 g, a cigar to 5 g and a pack of pipe tobacco to 50 g. Cumulative tobacco consumption was calculated for ever-smokers in pack-years, which was defined as smoking of 20 g of tobacco per day for a year. The participants were asked whether they had been exposed to dust and/or welding fumes at work. In the 1991–1994 examination, the participants were also asked whether any person in their household was a smoker or whether one of their parents used to be smokers. In the 2001–2003 examination, the participants were additionally asked whether they were
Data analyses were carried out using STATA/SE 11.1 (StataCorp LP, College Station, Texas, USA). We used the trend test and multiple regression. Odds ratios were calculated using logistic regression and adjusted for age and sex, or for age, sex and cumulative tobacco consumption; for the latter, missing data for cumulative tobacco consumption (n = 58) were imputed using age and sex. The P-values for interaction between genotype and cumulative tobacco consumption were calculated using a regression model where the simple model with lung function, genotype and cumulative tobacco consumption was nested in a model also containing a two-factor term (genotype cumulative tobacco consumption). Meta-analyses were carried out with our previous study [13] between the CHRNA3 genotype and lung function/COPD (Figs 4 and 5) using fixed and random-effect models. Mediation analysis was carried out using the CHRNA3 genotype as an independent variable, disease (COPD or lung function) as a dependent variable and cumulative tobacco consumption as a mediator variable. The indirect effect of genotype on lung function/COPD through cumulative tobacco consumption was determined using the product of the coefficients method.
In the present study, we included participants from the examinations in 1991–1994 and 2001–2003. Of 10 605 participants with available DNA, 261 (2.5%) participants were not of Danish descent and were therefore excluded from statistical analyses. For CYP3A5*3 and CHRNA3, the genotype was determined in 10 341 (5754 women and 4587 men) and 10 330 (5746 women and 4584 men) of a total 10 344 available participants with DNA. Genotyping
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
222 Pharmacogenetics and Genomics
2014, Vol 24 No 4
For CYP3A5*3, the most common allele among Whites is the G allele, and therefore, the CYP3A5*3 GG homozygous genotype group was used as the reference group when calculating risk estimates; however, the G allele is the allele with a splice defect and presumed lack of enzyme activity and therefore the AA genotype is called a noncarrier. For CHRNA3, the most common allele among Whites is the C allele, and therefore, the CHRNA3 CC homozygous genotype group was used as the reference group when calculating risk estimates.
Results We included a total of 10 330 participants with a known CHRNA3 genotype, of whom 44.6% were noncarriers, 44.3% were heterozygotes and 11.1% were homozygotes (Table 1). We included a total of 10 341 participants with a known CYP3A5*3 genotype, of whom 0.5% were noncarriers (homozygous for the rare functional allele in Whites), 13.1% were heterozygotes and 86.4% were homozygotes. These frequencies are comparable with those reported in previous studies of Whites [5] and both were in Hardy–Weinberg equilibrium (P = 0.79 for CHRNA3 and 0.37 for CYP3A5*3). Characteristics did not differ by genotype (Table 1). Information on detailed smoking behaviour and CHRNA3 genotype has been
Table 1
published previously, where an association was found with daily tobacco consumption (P = 0.0001), cumulative tobacco consumption (P = 0.003) and smoke inhalation (P = 0.001) [8]. Characteristics on detailed smoking behaviour for CYP3A5*3 did not differ by genotype (Supplementary Table 1; Supplemental digital content 1, http://links.lww.com/FPC/A705). Lung function
For CHRNA3, FEV1% predicted was 89.3, 90.6 and 92.4% in homozygous, heterozygous and noncarrier ever-smokers, respectively (P-trend < 0.001; Table 2). The corresponding values for FVC% predicted were 94.5, 95.2 and 96.7% (P-trend < 0.001), and for FEV1/FVC ratio, the values were 0.753, 0.760 and 0.764 (P-trend = 0.008), respectively. When adjusted for cumulative tobacco consumption, these associations remained significant. The results were very similar when adjusted for smoking status (current and former smokers instead of cumulative tobacco consumption; data not shown). No interaction was detected between genotype and cumulative tobacco consumption (all P > 0.05; Table 2). In never-smokers, FVC% predicted for CHRNA3 were 99.1, 99.6 and 100.5% in homozygous, heterozygous and noncarriers, respectively (P-trend = 0.03); however, this association disappeared when
Characteristics of participants by CHRNA3 and CYP3A5*3 genotypes in the Danish general population, the Copenhagen City Heart
Study
Characteristics
CHRNA3 (rs1051730)
CYP3A5*3 (rs776746)
Heterozygotes (CT)
Heterozygotes (AG)
Noncarriers (CC)
All participants [n (%)] 4611 (44.6) Women [n (%)] 2587 (56.1) Age (years) 59.0 (44.8–69.6) Ever-smokers [n (%)] 3471 (75.4) Exposure to dust and/or welding 860 (18.8) smoke at work [n (%)] Passive smoking [n (%)] 3633 (79.8)
4572 2562 59.5 3514 848
PHomozygotes (TT) trend Noncarriers (AA)
(44.3) 1147 (11.1) (56.0) 597 (52.0) (45.4–69.8) 59.2 (45.6–69.1) (77.0) 880 (76.8) (18.7) 249 (21.8)
3658 (80.8)
928 (82.1)
Homozygotes (GG)
58 (0.5) 1349 (13.1) 0.06 28 (48.3) 766 (56.8) 0.79 55.9 (37.3–68.6) 59.8 (45.6–69.3) 0.13 41 (70.7) 1014 (75.2) 0.11 11 (19.3) 250 (18.7)
8934 4960 59.2 6820 1697
0.06
7112 (80.7)
43 (74.1)
1064 (79.8)
(86.4) (55.5) (45.2–69.7) (76.5) (19.1)
Ptrend 0.71 0.52 0.17 0.75 0.26
Values are median (interquartile range) for age and number (%) for categorical variables. The P-values were calculated in a trend test.
Table 2 Lung function in ever-smokers by CHRNA3 and CYP3A5*3 genotypes in the Danish general population, the Copenhagen City Heart Study CHRNA3 (rs1051730)
FEV1% predicted FVC% predicted FEV1/FVC ratio
CYP3A5*3 (rs776746)
Noncarriers (CC) (N = 3412)
Heterozygotes (CT) (N = 3469)
Homozygotes (TT) (N = 869)
P-trend
P-value adjusted
P-value for interaction
Homozygotes (GG) (N = 6714)
Heterozygotes (AG) (N = 1005)
92.4±20.5
90.6±21.3
89.3±21.3
< 0.001
< 0.001
0.30
91.3±21.2
90.3±19.9
93.7±17.3
0.13
96.7±17.7
95.2±18.2
94.5±18.1
< 0.001
0.001
0.27
95.9±18.1
94.9±17.2
97.2±14.7
0.15
0.764±0.094
0.760±0.099
0.753±0.099
0.02
0.10
0.761±0.097
0.761±0.093
0.770±0.077
0.73
0.008
Noncarriers P(AA) (N = 41) trend
Data are mean±SD. P-trend was calculated with genotypes coded 0, 1 and 2. P-values adjusted were adjusted for cumulative tobacco consumption and calculated by multiple regression. P-values for interaction were between genotype and cumulative tobacco consumption. Because some individuals lacked information on spirometry, the numbers in this table and Table 3 do not add up to the total number of individuals genotyped, as mentioned in Table 1. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.
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CHRNA3 and CYP3A5*3 genotypes and lung disease Kaur-Knudsen et al. 223
adjusted for multiple comparison and thus no association was found between the CHRNA3 genotype and lung function in never-smokers (all P-trends > 0.05; Table 3). CYP3A5*3 did not associate with any lung function measurements in ever-smokers or never-smokers (all P-trends > 0.05; Tables 2 and 3).
Chronic obstructive pulmonary disease
The CHRNA3 genotype was associated with increased risk of COPD in ever-smokers from noncarriers to heterozygotes to homozygotes when adjusted for age and sex, irrespective of the definition used (P-trends = 0.02 to
