Association of Gc-globulin variation with susceptibility to COPD and diffuse panbronchiolitis

Chronic obstructive pulmonary disease (COPD) is now becoming the most prevalent respiratory disease, and it is estimated that COPD will be the fifth leading cause of the Disability-Adjusted Life Year in the world in 2020 1. Although the most prevalent risk factor for the development of COPD is tobacco smoking, only 10–20% of heavy smokers develop symptomatic COPD 2. Recent studies suggest that genetic factors, excluding α1-antitrypsin, might be related to the individual susceptibility to COPD 3, 4. Additionally, based on the protease-antiprotease hypothesis on the pathogenesis of COPD 5, neutrophils that produce neutrophil elastase have been thought to play a key role in the pathogenesis of COPD.

Diffuse panbronchiolitis (DPB), first described in 1969 6, is also characterized by chronic airflow limitation, 6, and the aetiology of the disease is still unknown. Cases of DPB are mainly found in East Asian populations 7, 8, and neither environmental factors nor infections specific to the disease have been demonstrated. It has also been reported that human leukocyte antigen-;B alleles or a closely linked gene contribute to genetic predisposition in DPB 9. After considering these facts, the authors hypothesized that genetic factors may be associated with the pathogenesis of DPB. The chemotactic activity of neutrophils is considered to be important in the pathogenesis of DPB because: 1) DPB is characterized by the infiltration of inflammatory leukocytes into the respiratory bronchioles; and 2) erythromycin, the first choice of therapy for DPB, was reported to inhibit the elastolytic-like activity 10 and chemotactic activity of neutrophils 10–12.

Gc-globulin (vitamin D-;binding protein) is known to bind with vitamin D and transport it. However, it has also been reported that Gc-globulin enhances the neutrophil chemotactic activity of complement component 5a (C5a), C5a without the carboxyl-terminal arginine (C5a des Arg) 13 and human monocyte chemotaxis by a different pathway 14. Gc-globulin exhibits considerable genetic variability due to its three polymorphic alleles (GC*1F, GC*1S, and GC*2) and >80 rarer alleles 15. Although several investigators have previously reported an association between this genetic variation and COPD, the conclusion is controversial 16–19. Low frequency for the GC*2/GC*2 genotype in COPD was observed in two reports at a serological level 16, 17, and in one report at a deoxyribonucleic acid (DNA) level 18. However, Kauffmann et al. 19 did not detect any association between this genotype and COPD. Although Horne et al. 17 reported a positive association between GC*1F allele and airway obstruction, others have not yet been able to confirm this result 16, 18, 19.

In this study, the authors investigated whether the three polymorphic alleles (GC*1F, GC*1S, and GC*2) of Gc-globulin are associated with the susceptibility of COPD and DPB by using a polymerase chain reaction (PCR)-based genotyping assay.

Material and methods

Methods for genotyping

The method used for genotyping Gc-globulin was modified from a previously described method 18. Briefly, genomic DNA was extracted from whole blood using the QIAamp Blood Kit (QIAGEN Inc., Valencia, CA, USA). PCR and restriction enzyme fragment length polymorphism assays were performed to genotype the three polymorphic alleles (GC*1F, GC*1S, and GC*2) of Gc-globulin.

The primer pair Gc-F1: 5′-;TAATGAGCAAATGAAAGAAG-;3′ and Gc-R1: 5′-;AATCACAGTAAAGAGGAGGT-;3′ were used for this assay. PCRs were carried out with a Programme-temp-control System PC-800 PCR machine (Astec, Tokyo, Japan). Genomic DNA (200 ng) was added to a 30-µL reaction mixture also containing 22 pmol of each primer and 1 U of AmpliTaq Gold (Perkin-Elmer Applied Biosystems Division, Foster City, CA, USA), in 1× PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.001% (per cent weight in volume (w/v)) gelatin and 0.2 mM of each deoxynucleotide triphosphate (dNTP). The “auto-hot start” was used to prevent nonspecific priming in the first cycle of PCR. Following an initial denaturation step at 95°C for 12 min, 40 cycles of PCR were carried out (denaturation at 95°C for 30 s, annealing at 50°C for 30 s, elongation at 72°C for 60 s), which were followed by one cycle of elongation at 72°C for 5 min. The PCR products were separated and each was digested with 10 U of Hae III (New England BioLabs Inc., Beverly, MA, USA) and 10 U of Sty I (New England BioLabs Inc.).

All the digests were electrophoresed on a gel containing 2.5% Ultrapure Agarose (GibcoBRL, Gaithersburg, MD, USA) and ethidium bromide. The film of the bands electrophoresed on the gel is shown in figure 1⇓.

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Fig. 1.—

Representative electrophoresis gel with band visualization in the Gc-globulin polymorphism. All six genotypes are shown. The first lane is 100–1,000 base pairs (bp) deoxyribonucleic acid (DNA) ladder (the difference between each band is 100 bp). H: cut by Hae III; S: cut by Sty I; 1F: GC*1F; 1S: GC*1S; 2: GC*2.

Results

The basic characteristics of the COPD and DPB groups were as follows: age (yr) was 68.3±9.9 versus 60.3±13.8; distribution of sex (male/female) was 60/3 versus 45/37; and FEV₁/FVC of pulmonary function tests was 44.3±12.7% versus 56.4±14.1%, respectively. Other results of the pulmonary function tests of the COPD group were as follows: RV was 2.56±0.11 L, TLC was 5.67±0.89 L, RV/TLC was 45.5±9.2%, D_L,CO was 11.0±3.4 mL·min⁻¹·mmHg⁻¹, and % D_L,CO was 49.2±14.0%. The average titre of cold haemagglutinin of the DPB group was 28.7±2.2. Smoking history (pack-yrs) of the COPD subjects was 102.2±40.4, and that of the control subjects was unknown because they were anonymous donors. The basic characteristics of COPD subjects are summarized in table 1⇓.

The distribution of the six major genotypes of Gc-globulin in the control, COPD and DPB groups are presented in table 2⇓. The proportion of GC*1F homozygotes was significantly higher in the COPD patients than in the control subjects (COPD 36.5% versus control 20.7%, p=0.0351), and the odds ratio (OR) for GC*1F homozygotes versus all other genotypes was 2.2 (95% CI 1.1–4.6) for the COPD group. The proportion with GC*1F was also significantly higher in the COPD patients (COPD 85.7% versus control 75.6%, p=0.0357), and the OR was 1.9 (95% CI 1.2–3.2). In addition, the lung function parameters (FEV₁/FVC, RV, TLC, RV/TLC, D_L,CO, and % D_L,CO) and smoking history between GC*1F homozygotes and those of the other genotypes were compared. However, no correlation between these parameters and GC*1F (data not shown) was detected.

The frequency of the three alleles is shown in table 3⇓. In the control group, the frequency of the GC*1F, GC*1S, and GC*2 allele was 0.48, 0.27, and 0.24, respectively. The present results, differing from those of Caucasians (frequency of GC*1F, GC*1S, GC*2; 0.19, 0.53, 0.28, respectively) 18, agree with those obtained in a previous study of a Japanese population 21. Because the distribution of the sex was different between the COPD (male/female, 60/3) and the control subjects (male/female, 42/40), the allele frequency between the male and female control subjects was compared. As shown in table 3⇓, the difference of the allele frequency between these two groups was not statistically significant (p=0.99). Therefore, it was reasonable to consider that the difference of sex could not influence the distribution of this genetic variation.

The authors did not find any association between distribution of the genotype and frequency of the three polymorphic alleles of Gc-globulin and DPB (tables 2⇑ and 3⇑).

Discussion

COPD and DPB are diseases characterized by chronic airflow limitation. Although the aetiology of these diseases is still under investigation, neutrophils have commonly been assumed to play a key role in their pathogenesis. Therefore, variation in genes with a function related to chemotaxis of neutrophils may confer a risk for the development of COPD and DPB. Since Gc-globulin has been reported to be associated with chemotaxis of neutrophils 13 and monocytes 14, the authors investigated the association between genetic variation that codes the 416th and 420th amino acid of Gc-globulin and susceptibility to COPD and DPB.

This study showed that the proportion of COPD patients with the GC*1F allele was significantly higher than that of control subjects with this allele. However, there was no difference in the allele frequency and distribution of genotypes of the Gc-globulin between the DPB and control groups.

Chronic tobacco smoking is the major risk factor for the development of COPD. However, only a small proportion of smokers develop airway limitation. The genetic factors are supposed to be related to this individual susceptibility. Recently, genetic polymorphism of cytochrome P450 22, microsomal epoxide hydrolase 23, glutathione S-;transferase M1 24, tumour necrosis factor-;α 25, and glutathione S-;transferase P1 26 were suggested to have an association with emphysema or COPD.

According to the results of this study, it is assumed that a genotype with the GC*1F allele might be one of the risk factors for COPD. Previously, the association between this genetic variation and COPD was reported by several studies. Kueppers et al. 16 have reported a lower frequency for the GC*2/GC*2 genotype in COPD (relative risk=0.2), and Horne et al. 17 have confirmed this data. However, in another study by Kauffmann et al. 19, no association was found, and the role of the GC*2 allele of Gc-globulin in the pathogenesis of COPD has remained controversial. All of these studies 16, 17, 19 were performed at a serological level. A recent study by Schellenberg et al. 18 was performed at a DNA level and reported the protective effect of the GC*2/GC*2 genotype for COPD (OR=0.17). However, the present study could not confirm this effect. It is possible that racial differences might have caused this discrepancy between results 18, 21, however, this remains to be elucidated.

These reports showed the common finding that the frequency of the GC*1F allele was higher in the COPD group 16–19, and one report also suggested that the homozygous GC*1F allele increased the risk of developing COPD 17, which was compatible with the present results.

The difference of the function of Gc-globulin between the GC*1F allele and the other alleles has not yet been elucidated. The different genotypes of Gc-globulin are known to have post-translational carbohydrate differences 27. Gc1 and Gc2 were reported to have different pathways of deglycosylation 28, and the deglycosylated form of Gc-globulin has been shown to be a potent macrophage-activating factor 27. Therefore, in addition to the possibility that Gc-globulin polymorphism modulates neutrophil and monocyte chemotaxis, the differences in deglycosylated forms of Gc1f, Gc1s, and Gc2 may effect activation of macrophages, whose metalloproteinase was recently reported to play a key role in the pathogenesis of emphysema 29.

Although the GC*2 allele has been reported to be associated with the chemotactic activity of neutrophils 13, this was not ascertained in a recent study 18, and the present authors found no association between the genotype with the GC*2 allele and the susceptibility to COPD.

In conclusion, it is suggested that the genotype the GC*1F allele of Gc-globulin might be one of the risk factors for chronic obstructive pulmonary disease. However, no association between this polymorphism of Gc-globulin and susceptibility to diffuse panbronchiolitis was found in the present study.