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ORIGINAL ARTICLE
Association of five genetic variants with chronic obstructive pulmonary disease susceptibility and spirometric phenotypes in a Chinese Han population JING YANG,1* HAIXIA ZHOU,1* BINMIAO LIANG,1 JUN XIAO,1 ZHIGUANG SU,2 HONG CHEN,1 CHUNLAN MA,1 DENGXUE LI,3 YULIN FENG1 AND XUEMEI OU1 1
Departments of Respiratory Medicine and 2Geriatrics, West China Hospital, Sichuan University, Chengdu and 3Department of Respiratory Medicine, The Second People’s Hospital of Hongya County, Meishan, Sichuan Province, China
ABSTRACT Background and objective: Recent genome-wide association studies have shown associations between variants at five loci (TNS1, GSTCD, HTR4, AGER and THSD4) and chronic obstructive pulmonary disease (COPD) or lung function. However, their association with COPD has not been proven in Chinese Han population, nor have COPD-related phenotypes been studied. The objective of this study was to look for associations between five single nucleotide polymorphisms (SNP) in these novel candidate genes and COPD susceptibility or lung function in a Chinese Han population. Methods: Allele and genotype data on 680 COPD patients and 687 healthy controls for sentinel SNP in these five loci were investigated. Allele frequencies and genotype distributions were compared between cases and controls, and odds ratios were calculated. Potential relationships between these SNP and COPD-related lung function were assessed. Results: No significant associations were found between any of the SNP and COPD in cases and controls. The SNP (rs3995090) in HTR4 was associated with COPD (adjusted P = 0.022) in never-smokers, and the SNP (rs2070600) in AGER was associated with forced expiratory volume in 1 s (FEV1%) predicted (β = −0.066, adjusted P = 0.016) and FEV1/forced vital capacity (β = −0.071, adjusted P = 0.009) in all subjects. Conclusions: The variant at HTR4 was associated with COPD in never-smokers, and the SNP in AGER was associated with pulmonary function in a Chinese Han population. Key words: AGER, chronic obstructive pulmonary disease, HTR4, pulmonary function, single-nucleotide polymorphism. Abbreviations: AGER, advanced glycation end-products receptor; BMI, body mass index; COPD, chronic obstructive Correspondence: Xuemei Ou, Department of Respiratory Medicine, West China Hospital, Sichuan University, 37# Guo-xuexiang, Chengdu 610041, Sichuan Province, China. Email: [email protected] *Contributed equally to this study. Received 21 May 2013; invited to revise 20 July 2013; revised 17 August 2013; accepted 12 September 2013 (Associate Editor: Robert Young). © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
SUMMARY AT A GLANCE We demonstrate that variants in HTR4 are associated with COPD in never-smokers, and SNP in AGER are associated with lung function in a Chinese Han population.
pulmonary disease; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; GSTCD, glutathione S-transferase C-terminal domain; GWAS, genome-wide association studies; HTR4, 5-hydroxytryptamine receptor-4; SNP, single nucleotide polymorphism; THSD4, type 1, domain-containing 4; TNS1, tensin 1.
INTRODUCTION Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is not fully reversible in the airways.1 It will rise from the fifth leading cause of death in 2002 to the third leading cause by 2030 worldwide,2 and the third in rural areas in China investigated in 2009.3 Although tobacco smoking is the most significant risk for COPD, not all smokers will develop the disease,4 and genetic factors also play important roles in the pathogenesis of COPD.5 Although multiple studies in diverse populations have demonstrated a large genetic contribution to COPD susceptibility6–10 or decline in lung function,11 to date few candidate genes have been identified convincingly.12,13 However, several candidate gene studies have obtained inconsistent results,14 which might be attributed to many uncertain reasons, including small sample size, environmental factors or ethnic diversity. A previous genome-wide association study (GWAS) has identified some genomic regions that are associated with COPD-related lung function in 20 288 participants of European ancestry.15 These regions are located in tensin 1 (TNS1) on chromosome 2q35, glutathione S-transferase C-terminal domain (GSTCD) on chromosome 4q24, 5-hydroxytryptamine receptor-4 (HTR4) on chromosome 5q33, advanced Respirology (2014) 19, 262–268 doi: 10.1111/resp.12212
263
Genetic variants with COPD
glycation end-products receptor (AGER) on chromosome 6p21 and thrombospondin, type 1, domaincontaining 4 (THSD4) on chromosome 15q23. In addition to this study testing lung function as an outcome, a recent large-scale GWAS meta-analysis demonstrated significant association of these genomic regions with COPD susceptibility other than lung function in Europeans.16 Given the large differences in genetic background of different ethnic populations, studies in other populations are warranted. We hypothesized that variants of these genes might be associated with the occurrence and development of COPD or reduction in lung function in a Chinese Han population. To test this hypothesis, we genotyped 1367 individuals from the adult southwestern Chinese Han population, and examined associations among allele, genotype, lung function and risk of COPD. Moreover, as we hoped to know whether the effect of these gene variants on COPD susceptibility was associated with smoking behaviour, smoking stratified analysis between single nucleotide polymorphism (SNP) and COPD susceptibility was taken in this study.
METHODS Participants In total, 680 COPD patients and 687 healthy controls were recruited from a southwestern Chinese Han population. All subjects provided written informed consent. This study was approved by the ethics committee of West China Hospital of Sichuan University. COPD was diagnosed according to the criteria established by the Global Initiative for Chronic Obstructive Lung Disease:1 age ≥ 40 years, airflow limitation as indicated by post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) of ≤70% and FEV1 of ≤80% predicted. Controls were age ≥ 40 years old and had normal lung function (post-bronchodilator FEV1 > 80% predicted and FEV1/ FVC > 0.7). All subjects were excluded if they had a comorbidity diagnosis of asthma or lung cancer, or had other significant respiratory diseases or any hereditary diseases. Controls were also excluded if they had family history of COPD. Efforts were made to match cases by age, gender, body mass index (BMI) and smoking history.
SNP selection and genotyping We chose sentinel SNP at each of the following loci: TNS1 (rs2571445), GSTCD (rs10516526), HTR4 (rs39 95090), AGER (rs2070600) and THSD4 (rs12899618). The SNP in these five genes were found to be significantly associated with lung function and the risk of COPD in a non-Asian population by recent GWAS.15,16 The characteristics of these SNP were obtained by searching the dbSNP database of NCBI (http:// www.ncbi.nlm.nih.gov/). A 5-mL peripheral blood sample was obtained from each participant for DNA analysis. We extracted genomic DNA using a commercially available extrac© 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
tion kit (Tiangen Biotech Co., LTD, Beijing, China). Genotyping was undertaken commercially by the Beijing Genomics Institute (Shenzhen, China) as described,17 using Sequenom’s iPLEX SNP genotyping protocol developed for measurement with the MassARRAY mass spectrometer (Sequenom, San Diego, CA, USA).18 As a quality control measure, 5% of samples were genotyped in duplicate to check for concordance. In addition, some samples were genotyped using restriction enzyme digestion or direct sequencing to confirm the genotyping results from Beijing Genomics Institute. Genotyping was done blinded to case or control status of samples.
Statistical analysis All demographic data were analyzed using the SPSS 18.0 software package (SPSS Inc., Chicago, IL, USA). Continuous variables (age, BMI, pack-years and pulmonary function) were calculated and presented as means (± standard deviation), and the two-tailed Student’s t-test was used to determine significant differences in these clinical data between the COPD cases and the controls. Categorical variables (gender and smoking status) are presented as number (percentage), and Pearson’s χ2 test was used to evaluate the difference. The χ2 test and multinomial logistic regression were applied to compare allele and genotype frequencies between cases and controls. Logistic analysis was adjusted for age, gender, BMI and pack-years. In order to exclude the impact of smoking behaviour on the association analysis, both case and control groups were each split into equal tertiles of smoking status (never-smoker and ever-smoker). In each tertile, the relationship between SNP and COPD was tested with logistic regression adjusted by the potential confounders given above. The relative risk associated was estimated as an odds ratio with a 95% confidence interval. In addition, linear regression was performed to assess the association between SNP and COPDrelated lung function, and to control for age, gender, BMI and pack-years of smoking. Each SNP in the controls was tested for deviation from the HardyWeinberg equilibrium using a goodness-of-fit χ2 test. The significance level for all tests was two-side value of P < 0.05.
RESULTS Study population characteristics The clinical and functional characteristics of COPD patients and control subjects are shown in Table 1. They did not significantly differ in gender, BMI or smoking status. Differences between age and packyears for ever-smokers in two groups were observed (P = 0.002 and P = 0.001, respectively). As expected, FEV1% predicted, FVC% predicted and FEV1/FVC of the cases were decreased significantly compared with the controls (P < 0.001, P = 0.017 and P < 0.001, respectively). Respirology (2014) 19, 262–268
264
J Yang et al.
Allele and genotype frequencies of SNP in five loci The allele frequencies for five SNP in all participants are shown in Table 2. One SNP (rs10516526) in GSTCD was not found in COPD patients or control subjects, so this SNP was removed from the study. There was no deviation from Hardy-Weinberg equilibrium for any genotyped SNP in the controls, and none of these SNP showed significant association with COPD in the unadjusted analysis. For analysis of genotypic association of these SNP with COPD (Table 3), there were no significant differences between two groups even after adjusting the analysis for age, gender, BMI and pack-years. As tobacco smoking plays a key role in COPD, and we carried out a smoking status stratification analysis to eliminate any potential confounding caused by the difference in smoking behaviour and to illuminate the potential relationships between these SNP with COPD (Table 4). One SNP (rs3995090) in
Table 1
Description of cases and controls
Item n Age (years) Male (%) BMI (kg/m2) Smoking status Never-smoker (%) Ever-smoker (%) Pack-years for ever-smokers FEV1% predicted (%) FVC% predicted (%) FEV1/FVC
Cases
Controls
P-value
680 62.7 (9.1) 483 (71.0) 22.5 (3.5)
687 60.9 (10.2) 476 (69.3) 22.6 (3.3)
0.002† 0.481‡ 0.085†
225 (33.1) 455 (66.9) 33.8 (20.7)
231 (33.6) 456 (66.4) 29.2 (21.3)
0.001†
64.2 (24.9) 98.3 (41.5) 53.4 (12.5)
102.4 (15.6) 103.3 (18.1) 79.3 (6.1)
ORIGINAL ARTICLE
Association of five genetic variants with chronic obstructive pulmonary disease susceptibility and spirometric phenotypes in a Chinese Han population JING YANG,1* HAIXIA ZHOU,1* BINMIAO LIANG,1 JUN XIAO,1 ZHIGUANG SU,2 HONG CHEN,1 CHUNLAN MA,1 DENGXUE LI,3 YULIN FENG1 AND XUEMEI OU1 1
Departments of Respiratory Medicine and 2Geriatrics, West China Hospital, Sichuan University, Chengdu and 3Department of Respiratory Medicine, The Second People’s Hospital of Hongya County, Meishan, Sichuan Province, China
ABSTRACT Background and objective: Recent genome-wide association studies have shown associations between variants at five loci (TNS1, GSTCD, HTR4, AGER and THSD4) and chronic obstructive pulmonary disease (COPD) or lung function. However, their association with COPD has not been proven in Chinese Han population, nor have COPD-related phenotypes been studied. The objective of this study was to look for associations between five single nucleotide polymorphisms (SNP) in these novel candidate genes and COPD susceptibility or lung function in a Chinese Han population. Methods: Allele and genotype data on 680 COPD patients and 687 healthy controls for sentinel SNP in these five loci were investigated. Allele frequencies and genotype distributions were compared between cases and controls, and odds ratios were calculated. Potential relationships between these SNP and COPD-related lung function were assessed. Results: No significant associations were found between any of the SNP and COPD in cases and controls. The SNP (rs3995090) in HTR4 was associated with COPD (adjusted P = 0.022) in never-smokers, and the SNP (rs2070600) in AGER was associated with forced expiratory volume in 1 s (FEV1%) predicted (β = −0.066, adjusted P = 0.016) and FEV1/forced vital capacity (β = −0.071, adjusted P = 0.009) in all subjects. Conclusions: The variant at HTR4 was associated with COPD in never-smokers, and the SNP in AGER was associated with pulmonary function in a Chinese Han population. Key words: AGER, chronic obstructive pulmonary disease, HTR4, pulmonary function, single-nucleotide polymorphism. Abbreviations: AGER, advanced glycation end-products receptor; BMI, body mass index; COPD, chronic obstructive Correspondence: Xuemei Ou, Department of Respiratory Medicine, West China Hospital, Sichuan University, 37# Guo-xuexiang, Chengdu 610041, Sichuan Province, China. Email: [email protected] *Contributed equally to this study. Received 21 May 2013; invited to revise 20 July 2013; revised 17 August 2013; accepted 12 September 2013 (Associate Editor: Robert Young). © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
SUMMARY AT A GLANCE We demonstrate that variants in HTR4 are associated with COPD in never-smokers, and SNP in AGER are associated with lung function in a Chinese Han population.
pulmonary disease; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; GSTCD, glutathione S-transferase C-terminal domain; GWAS, genome-wide association studies; HTR4, 5-hydroxytryptamine receptor-4; SNP, single nucleotide polymorphism; THSD4, type 1, domain-containing 4; TNS1, tensin 1.
INTRODUCTION Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is not fully reversible in the airways.1 It will rise from the fifth leading cause of death in 2002 to the third leading cause by 2030 worldwide,2 and the third in rural areas in China investigated in 2009.3 Although tobacco smoking is the most significant risk for COPD, not all smokers will develop the disease,4 and genetic factors also play important roles in the pathogenesis of COPD.5 Although multiple studies in diverse populations have demonstrated a large genetic contribution to COPD susceptibility6–10 or decline in lung function,11 to date few candidate genes have been identified convincingly.12,13 However, several candidate gene studies have obtained inconsistent results,14 which might be attributed to many uncertain reasons, including small sample size, environmental factors or ethnic diversity. A previous genome-wide association study (GWAS) has identified some genomic regions that are associated with COPD-related lung function in 20 288 participants of European ancestry.15 These regions are located in tensin 1 (TNS1) on chromosome 2q35, glutathione S-transferase C-terminal domain (GSTCD) on chromosome 4q24, 5-hydroxytryptamine receptor-4 (HTR4) on chromosome 5q33, advanced Respirology (2014) 19, 262–268 doi: 10.1111/resp.12212
263
Genetic variants with COPD
glycation end-products receptor (AGER) on chromosome 6p21 and thrombospondin, type 1, domaincontaining 4 (THSD4) on chromosome 15q23. In addition to this study testing lung function as an outcome, a recent large-scale GWAS meta-analysis demonstrated significant association of these genomic regions with COPD susceptibility other than lung function in Europeans.16 Given the large differences in genetic background of different ethnic populations, studies in other populations are warranted. We hypothesized that variants of these genes might be associated with the occurrence and development of COPD or reduction in lung function in a Chinese Han population. To test this hypothesis, we genotyped 1367 individuals from the adult southwestern Chinese Han population, and examined associations among allele, genotype, lung function and risk of COPD. Moreover, as we hoped to know whether the effect of these gene variants on COPD susceptibility was associated with smoking behaviour, smoking stratified analysis between single nucleotide polymorphism (SNP) and COPD susceptibility was taken in this study.
METHODS Participants In total, 680 COPD patients and 687 healthy controls were recruited from a southwestern Chinese Han population. All subjects provided written informed consent. This study was approved by the ethics committee of West China Hospital of Sichuan University. COPD was diagnosed according to the criteria established by the Global Initiative for Chronic Obstructive Lung Disease:1 age ≥ 40 years, airflow limitation as indicated by post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) of ≤70% and FEV1 of ≤80% predicted. Controls were age ≥ 40 years old and had normal lung function (post-bronchodilator FEV1 > 80% predicted and FEV1/ FVC > 0.7). All subjects were excluded if they had a comorbidity diagnosis of asthma or lung cancer, or had other significant respiratory diseases or any hereditary diseases. Controls were also excluded if they had family history of COPD. Efforts were made to match cases by age, gender, body mass index (BMI) and smoking history.
SNP selection and genotyping We chose sentinel SNP at each of the following loci: TNS1 (rs2571445), GSTCD (rs10516526), HTR4 (rs39 95090), AGER (rs2070600) and THSD4 (rs12899618). The SNP in these five genes were found to be significantly associated with lung function and the risk of COPD in a non-Asian population by recent GWAS.15,16 The characteristics of these SNP were obtained by searching the dbSNP database of NCBI (http:// www.ncbi.nlm.nih.gov/). A 5-mL peripheral blood sample was obtained from each participant for DNA analysis. We extracted genomic DNA using a commercially available extrac© 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
tion kit (Tiangen Biotech Co., LTD, Beijing, China). Genotyping was undertaken commercially by the Beijing Genomics Institute (Shenzhen, China) as described,17 using Sequenom’s iPLEX SNP genotyping protocol developed for measurement with the MassARRAY mass spectrometer (Sequenom, San Diego, CA, USA).18 As a quality control measure, 5% of samples were genotyped in duplicate to check for concordance. In addition, some samples were genotyped using restriction enzyme digestion or direct sequencing to confirm the genotyping results from Beijing Genomics Institute. Genotyping was done blinded to case or control status of samples.
Statistical analysis All demographic data were analyzed using the SPSS 18.0 software package (SPSS Inc., Chicago, IL, USA). Continuous variables (age, BMI, pack-years and pulmonary function) were calculated and presented as means (± standard deviation), and the two-tailed Student’s t-test was used to determine significant differences in these clinical data between the COPD cases and the controls. Categorical variables (gender and smoking status) are presented as number (percentage), and Pearson’s χ2 test was used to evaluate the difference. The χ2 test and multinomial logistic regression were applied to compare allele and genotype frequencies between cases and controls. Logistic analysis was adjusted for age, gender, BMI and pack-years. In order to exclude the impact of smoking behaviour on the association analysis, both case and control groups were each split into equal tertiles of smoking status (never-smoker and ever-smoker). In each tertile, the relationship between SNP and COPD was tested with logistic regression adjusted by the potential confounders given above. The relative risk associated was estimated as an odds ratio with a 95% confidence interval. In addition, linear regression was performed to assess the association between SNP and COPDrelated lung function, and to control for age, gender, BMI and pack-years of smoking. Each SNP in the controls was tested for deviation from the HardyWeinberg equilibrium using a goodness-of-fit χ2 test. The significance level for all tests was two-side value of P < 0.05.
RESULTS Study population characteristics The clinical and functional characteristics of COPD patients and control subjects are shown in Table 1. They did not significantly differ in gender, BMI or smoking status. Differences between age and packyears for ever-smokers in two groups were observed (P = 0.002 and P = 0.001, respectively). As expected, FEV1% predicted, FVC% predicted and FEV1/FVC of the cases were decreased significantly compared with the controls (P < 0.001, P = 0.017 and P < 0.001, respectively). Respirology (2014) 19, 262–268
264
J Yang et al.
Allele and genotype frequencies of SNP in five loci The allele frequencies for five SNP in all participants are shown in Table 2. One SNP (rs10516526) in GSTCD was not found in COPD patients or control subjects, so this SNP was removed from the study. There was no deviation from Hardy-Weinberg equilibrium for any genotyped SNP in the controls, and none of these SNP showed significant association with COPD in the unadjusted analysis. For analysis of genotypic association of these SNP with COPD (Table 3), there were no significant differences between two groups even after adjusting the analysis for age, gender, BMI and pack-years. As tobacco smoking plays a key role in COPD, and we carried out a smoking status stratification analysis to eliminate any potential confounding caused by the difference in smoking behaviour and to illuminate the potential relationships between these SNP with COPD (Table 4). One SNP (rs3995090) in
Table 1
Description of cases and controls
Item n Age (years) Male (%) BMI (kg/m2) Smoking status Never-smoker (%) Ever-smoker (%) Pack-years for ever-smokers FEV1% predicted (%) FVC% predicted (%) FEV1/FVC
Cases
Controls
P-value
680 62.7 (9.1) 483 (71.0) 22.5 (3.5)
687 60.9 (10.2) 476 (69.3) 22.6 (3.3)
0.002† 0.481‡ 0.085†
225 (33.1) 455 (66.9) 33.8 (20.7)
231 (33.6) 456 (66.4) 29.2 (21.3)
0.001†
64.2 (24.9) 98.3 (41.5) 53.4 (12.5)
102.4 (15.6) 103.3 (18.1) 79.3 (6.1)
