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Introduction
The age-dependent increase in the prevalence of chronic obstructive pulmonary disease (COPD) has suggested a relationship between the aging process and COPD development1,2. Although this interrelationship remains incompletely understood, aging and COPD share several similarities, including cardiovascular mortality, obstructive pulmonary physiology, and enlargement of airspaces3. In addition, inflammation associated with aging and COPD have several similarities, including neutrophil accumulation, NF-κB activation, and increased expression of markers of oxidative stress4.
Furthermore, the expression of antiaging molecules, such as sirtuins (SIRT), histone deacetylase (HDAC), senescence marker protein-30 (SMP30), and klotho, are reduced in COPD4. Among these, the klotho gene was incidentally discovered during the development of a hypertensive transgenic murine model5. The klotho protein has several functions, including the regulation of several growth factor signaling pathways, including those associated with fibroblast growth factor-23, insulin/insulin-like growth factor and Wnt, its activity in multiple ion channels, and its suppression of oxidative stress6. Homozygous mutant klotho (KL-/-) mice are regarded as a good animal model of human aging, since these mice have a short life span and exhibit multiple disorders resembling human premature-aging syndrome. Moreover, they exhibit emphysematous changes, characterized by the enlargement of air spaces distal to the terminal bronchioles, accompanied by the destruction of normal architecture5,6.
To our knowledge, the association between klotho gene polymorphisms and COPD has not been determined. We therefore performed genotype analyses of 2 klotho gene SNPs, G-395A in the promoter region and C1818T in exon 4, in Korean COPD patients and control subjects.
Materials and Methods
1. Subjects
Individuals with COPD were recruited, beginning in June 2005, from those in the "The Korean Obstructive Lung Disease cohort", a longitudinal prospective study of COPD in patients from the pulmonary clinics of 11 hospitals in Korea7. All COPD subjects included in this study had post-bronchodilator forced expiratory volume in one second [FEV1]/forced vital capacity [FVC] values of <0.7 and had more than 10 pack-years of smoking history. Complete CT scanning data, blood and other clinical information were obtained from all patients. All pulmonary function tests were performed as recommended by the American Thoracic Society/European Respiratory Society. Emphysema severity was measured quantitatively by determining the volume fraction of the lung below -950 Hounsfield units. Control subjects consisted of 305 smokers or ex-smokers with normal lung function registered in the Korean Genome Epidemiology Study (KoGES)8.
2. Genotyping
Genomic DNA was prepared from blood of all patients, and SNPs (rs1207568 and rs564481) were genotyped by the TaqMan method using ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA).
3. Statistics
The association between COPD susceptibility and SNP genotype was tested using logistic regression, adjusting for age, gender, and smoking pack-years, assuming an additive genetic model. The associations between emphysema severity and SNP genotypes were tested using linear regression, after adjusting for age, gender, and smoking pack-years, assuming an additive genetic model in Korean obstructive lung disease (KOLD) subjects. All statistical analyses were performed using SAS (SAS Institute, Cary, NC).
Results
1. Demographics characteristics of KOLD subjects
We assessed 219 subjects with COPD (mean age, 66.5 years) and 305 control subjects (mean age, 60.5 years) registered in the KOLD study. The mean FEV1 of the COPD subjects was 1.46 L (Table 1).
2. Genetic association with COPD susceptibility in the KOLD study
There were no genetic associations between either SNP and COPD susceptibility (p=0.61 and 0.20) (Table 2).
Discussion
We observed no associations between klotho gene polymorphisms and COPD susceptibility in subjects with COPD and controls. In addition, we found no association between klotho gene polymorphisms and emphysema severity in COPD subjects.
Accelerated aging of the lungs has recently been suggested as a pathogenic mechanism in COPD. Aging and emphysematous lungs have many similarities, including structural changes, with premature aging enhancing susceptibility to develop emphysema. However, differences in the extent of alveolar destruction have been observed in aging and emphysematous lungs9,10. To elucidate the mechanisms of aging and emphysema, senescence-accelerated mice, which possess mutant klotho genes and the senescence marker protein-30, have been utilized as an animal model. The klotho gene has been related to premature aging, a shorter life span, arteriosclerosis and osteoporosis, and has shown effects on air space enlargement. However, the association between the klotho gene and alveolar destruction is unclear11-13.
The klotho gene maps to chromosome 13q12 and several studies have assessed the relationships between klotho gene polymorphisms and the aging process. For example, the KL-VS allele of the klotho gene, containing a functional variant, has been found in Bohemian Czechs, Caucasians and African-Americans, and has shown an association with age-related phenotypes, including coronary artery disease, and blood pressure14-16. In an Italian population, the klotho KL-VS allele was not associated with longevity, although another Italian group reported that the klotho gene was associated with longevity, but only during a specific time period17,18.
To date, the KL-VS variant has not been observed in Asian subjects19. Rather, the polymorphisms G-395A and C1818T were associated with coronary artery disease20, stroke in women21,22, and hypertension23,24. Because these SNPs may be related to aging in Asian subjects, we investigated the association between COPD and these polymorphisms. In contrast to findings in cardiovascular diseases, we found no association between these SNPs and COPD. Since aging of the lungs involves a process similar to that of emphysema, we evaluated the relationship between emphysema severity and klotho genetic polymorphisms in the KOLD cohort, for which emphysema severity had been assessed using CT. However, we found no association between emphysema severity and klotho genetic polymorphisms.
This study had several limitations. First, both COPD cohorts consisted of mostly males. The genetic association of the klotho gene with cardiovascular risk in Koreans was observed only in females21,22, suggesting that the klotho gene may interact with gender. Because our cohort included mostly male subjects, any association in female subjects may not have been detected. Given that there is evidence of gender differences in emphysema25, the association of the klotho gene and emphysema should be studied separately in women.
A second limitation was that the numbers of subjects were relatively small. A third limitation is that we analyzed only two SNPs and we did not genotype the KL-VS variant. However, other studies in Asians have failed to detect the KL-VS variant, and the two genotyped SNPs have been associated with aging phenotypes in other studies. Lastly, our COPD subjects were recruited from multiple centers with different CT scanners that may influence the association. However, adjusting for center effect did not alter the results of association analysis.
In conclusion, the klotho gene was not associated with COPD susceptibility or emphysema severity in Korean subjects with COPD.
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