Chronic obstructive pulmonary disease (COPD) is a disease characterized by airflow limitation that is not fully reversible, but progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases.1 It is one of the leading causes of death worldwide, with increasing prevalence, mortality and economic burden.2 One interpretation of the pathogenesis of COPD is oxidant/antioxidant theory, which posits that oxidative stress initiates the onset of COPD whereas some antioxidant enzymes, such as heme oxygenase-1 (HOX-1) play a protective role in the lung.3,4 Recent studies have proposed that susceptibility to COPD might be related to the length, number, of (GT)n in the 5′-flanking region of HOX-1 gene and that smokers carrying the class L allele (n≥32) may have a higher risk for COPD.5 HOX-1 + alveolar macrophages were decreased in severe COPD.6 However, there has been no research about the relationship between severity of COPD and the polymorphism of HOX-1 gene.
Heme oxygenase, an essential enzyme in heme catabolism, cleaves heme to form biliverdin, which is subsequently converted to bilirubin by biliverdin reductase, and carbon monoxide, a putative neurotransmitter.7 Heme oxygenase activity is induced by its substrate heme and by various nonheme substances.8 Heme oxygenase occurs as two isozymes: an inducible heme oxygenase-1 and a constitutive heme oxygenase-2. HOX-1 is a key enzyme in heme catabolism that has been found to provide cellular protection against oxidant mediated, cellular injury because heme degradation products have antioxidant activity.9 The (GT)n dinucleotide repeat in the 5′-flanking region of HOX-1 gene shows length polymorphism and has been demonstrated to modulate gene transcription under thermal stress10 and associate with susceptibility to oxidant induced apoptosis in lymphoblastoid cell lines.11 It has been shown that (GT)n repeat is associated with emphysema susceptibility induced by cigarette smoking in Japanese population.5 We recently found that smokers with L allele were significantly higher with COPD than smokers without COPD (the author's unpublished data). These studies suggested that the 5′-flanking polymorphism in the HOX-1 gene is associated with the development of COPD. However, the association between the length of repeating polymorphism and severity of COPD is still unclear.
Therefore, we analysed these polymorphisms of HOX-1 in 452 patients at different stages of COPD, and tested whether the polymorphisms of HOX-1 influence the severity of COPD in Han population from Southwest China.
We selected 452 smokers with COPD who were recruited from the First Affiliated Hospital of Kunming Medical College (Kunming, China) and belonged to Han ethnic group from Southwest China. Smoking history was calculated by Brinkman's index (BI), that is, the number of cigarettes/day × the number of years. Informed consent for this study was obtained from all individuals and our institutional ethics committee approved this study.
All volunteers were diagnosed based on medical history, chest radiographic findings, physical examination and spirometric data. COPD was defined as the symptoms of cough, sputum production, or dyspnea, and/or a history of exposure to risk factors for the disease. The diagnosis was confirmed by spirometry. The presence of a post bronchodilator forced expiratory volume in one second (FEV1) < 80% of the predicted value in combination with an FEV1/forced vital capacity (FVC) < 70% confirmed the presence of airflow limitation that is not fully reversible (Table 1).
Genomic DNA was extracted using phenol- chloroform method from whole peripheral blood leukocytes collected into ethylenediamine tetraacetic acid (EDTA) tubes.12
Fragment analysis for HOX-1
The 5′-flanking region containing (GT)n repeats in HOX-1 gene was amplified by polymerase chain reaction (PCR) with a fluorescently labelled dCTP (Applied Biosyetems Inc., USA), a paired primer (sense primer: 5′-ACGCCTGGGGTGCATCAAGTC-3′; antisense primer: 5′-GTGGGGTGGAGAGGAGCAGTCATA-3′) 100 ng each other, 200 mmol/L dNTP and IU (20 mmol/L) Taq DNA polymerase (TaKaRa, Dalian, China). The PCR conditions in the thermal cycler (Eppendorf, Germany) was 30 cycles consisting of 94°C for 1 minute, 63°C for 1 minute and 72C for 1 minute. The sizes of the PCR products were determined by ABI PRISM 377 sequencer (Applied Biosyetems Inc.). In most cases, a blood sample had two different sizes of (GT)n repeats from different alleles. Each repeat number was calculated with Fragment Manager, version 1.2 (Pharmacia), with, as size markers, five cloned alleles that were loaded into every fourth lane in the DNA sequencer. The repeat numbers of these cloned alleles used as size markers were 16, 21, 31, 36 and 41(Fig. 1).
Pulmonary function testing
Pulmonary function tests were performed by the same type of electrospirometer (Autospiro AS-600, Minato Medical Science, Japan) used at the COPD clinic. In patients with COPD, the measurements were performed in their stable state, and there were no exacerbations of the conditions from the preceding 6 weeks.13 The use of bronchodilators was prohibited for at least 12 hours before the tests were performed. Based on the Global Initiative for Chronic Obstructive Lung Disease(GOLD),14 all COPD patients were classified into one of four stages: stage I (mild COPD: FEV1/FVC <0.70 and FEV1 ≥ 80% predicted); stage II (moderate COPD: FEV1/FVC < 0.70 and FEV1 <80% predicted and FEV1 ≥ 50% predicted); stage III (severe COPD: FEV1/FVC < 0.70 and FEV1 < 50% predicted and FEV1 ≥ 30% predicted); stage IV (very severe COPD: FEV1/FVC < 0.70 and FEV1 < 30% predicted or FEV1<50% predicted plus chronic respiratory failure).
Differences in clinical data (age, mean BI, FEV1% predicted and FEV1/FVC) of patients with COPD were compared by analysis of variance and sex data by chi-square test. If the analysis of variance was significant, then each mean was compared with each other using Student Newman Keuls method. The frequencies of genotypes of HOX-1 gene among different stages of COPD patients were compared by two tailed chi-square test. Statistical significance was defined as P<0.05. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to quantitatively assess the degree of association observed. SPSS v13.0 was used for all calculations.
The baseline characteristics and the results of baseline pulmonary function tests of 452 patients with COPD are shown in Table 1. In the evaluation of patients with COPD, 103 patients were classified in stage I, 112 in stage II, 115 in stage III, and 122 in stage IV. There was no significant difference in sex, age or smoking history (mean BI) between Groups of patients, but there were significant differences in the baseline FEV1 percentage predicted and FEV1/FVC among different stages of COPD. Moreover, comparison of means of the FEV1 predicted and FEV1/FVC between two stages by SNK, the differences were significant (P<0.05).
Genotyping and stages of COPD
HOX-1 genotype frequencies in different stage of COPD are shown in Table 2. The number of (GT)n repeats in HOX-1 gene showed a range of n = 10 to n = 39 and a trimodal distribution with three peaks located at 22, 29 and 33 repeats (Fig. 2). According to the number of (GT)n repeats, we divided the alleles into three subclasses: class S (≥25 GT repeats), class M (26–31 GT repeats) and class L (≥32 GT repeats). Furthermore, according to their HOX-1 genotypes, we divided the subjects into two groups: group I were individuals with class L allele, and group II were those without class L allele.
The genotypic frequency of Group I was significantly higher than group II in patients with very severe COPD (36.8% vs 22.4%, P<0.01, OR=2.0, 95% CI 1.3–3.1).
However, the genotypic frequency of the group I was lower in the mild COPD (16.0% vs 26.0 %, P=0.02, OR=0.5, 95% CI 0.3–0.9). In moderate and severe stages of COPD, there were similar proportions of the genotypic frequencies of HOX-1 group I and group II.
We investigated polymorphisms of HOX-1 gene in relation to the GOLD classification of COPD in a sample of Han nationality smokers from Southwest China. The main findings were that polymorphisms of HOX-1 gene were associated with severity of COPD: the genotypic frequency of Group I of HOX-1 gene was significantly higher in very severe COPD patients, in contrast, the genotypic frequency of the group I was lower in the mild COPD patients.
To our knowledge, there have been few articles analysing the relationship between the HOX-1 polymorphisms and COPD. Moreover, previous studies have shown varied results in association of polymorphism of HOX-1 and the risk of COPD. Firstly, Yamada et al5 showed a significant relationship between the polymorphisms of HOX-1 gene and development of COPD. Secondly, HOX-1 gene promoter polymorphisms may reduce the inducibility of HOX-1 by reactive oxygen species, thereby resulting in different susceptibility to damage by cigarette smoking. However, they could not verify the results shown by He, Budhi, Hersh and coworkers15–17 that no association between HOX-1 gene polymorphism and COPD. These studies were based on Japanese and Canadian populations, in which the frequencies of HOX-1 group I were 38% for Yamada,5 13.9% for He15 and 87% for Budhi and colleague's study.16 Our findings confirmed Yamada and coworkers' results5 in part although the frequency of the HOX-1 group I was slightly lower (31.9% vs 38%). Moreover, our study showed that the frequency for group I was increased gradually from stage I to stage IV. Therefore, we speculate that the promoter activity in HOX-1 gene may be modulated by the length variability of the (GT)n repeats and people with L allele tend to show a defective or weaker detoxifying capability in the lungs by decreasing the promoter activity in HOX-1 gene. As a result, the smokers carrying the class L allele may have a higher risk for very severe COPD, while HOX-1 II group genotype appears to function as a protective factor against the development of more sever COPD. Regretfully, a limitation of this study is the absence of a direct measure of activity of HOX-1. It is not, therefore, possible to clarify whether our results are due to relatively increased (GT)n repeats or to decreased activity of HOX-1.
The severity of COPD may be associated with age, cigarette consumption and sensitivity to effects of smoking.18 Our study showed that the age and smoking history of patients in different stages of COPD were more or less the same. However, in previous studies,5,15–17 no data were shown in match for age or smoking history. In addition, the discrepancy might also be due to different environmental factors and contribution of genes to lung function.19
It is more important to determine whether polymorphisms are associated with COPD severity because once a subset of patients is identified it may be possible to target more aggressive and effective therapeutic approaches to that group.20 Recently, the role of microsatellite polymorphism of HOX-1 gene promoter has been reported for some human diseases. It was shown that longer (GT)n repeat was associated with angiographic restenosis after coronary stenting,21 lung adenocarcinoma22 and pneumonia.23 These findings suggested that the polymorphism of the HOX-1 gene is associated with the strength of antiapoptotic effects of HOX-1, resulting in an association with susceptibility to oxidative stress mediated diseases. Our study confirmed that viewpoint in part. As a complex polygenic disease, it seemed that the mechanisms of COPD are influenced by actions of multiple genes and the genetic susceptibility may depend on the coincidence of several polymorphisms acting together.24 Thus, we speculated that persons with HOX-1 group I might be at a higher risk of developing very severe COPD while individuals with HOX-1 group II may be better able to protect their lung from more oxygenic injury caused by cigarette smoking and be more easily stabilized on mild COPD.
In conclusion, this study shows interactions between HOX-1 polymorphism and severity of COPD. We demonstrated the HOX-1 group I gene was associated with very severe COPD in a Southwest Chinese population in that COPD with the L allele of HOX-1 gene may be susceptible to a more severe COPD. Further studies of large size in various ethnic populations, as well as family studies, are indispensable to clarify the underlying molecular and pathophysiological mechanisms in the development of COPD.
We are grateful to all the donors who made this research possible.
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Keywords:© 2007 Chinese Medical Association
chronic obstructive pulmonary disease; heme oxygenase; polymorphism