Abstract
Mannose-binding lectin (MBL) insufficiency due to polymorphisms in the MBL2 gene causes an opsonization defect, which has been connected to infections and atopy. We investigated the significance of MBL2 genotypes with regard to persistent asthma and atopy among adults. The genotypes were determined in 243 adults with persistent asthma and 400 controls. Atopy was determined by skin-prick test. As a result, the carriage of −221 base pairs (bp) promoter region variant allele X (nucleotide change G→C; alleles Y→X, respectively) causing low MBL expression proved to be a significant risk factor for asthma in non-atopic males [odds ratio (OR) = 2·52, 95% confidence interval (CI) = 1·23–5·15; P = 0·01]. Furthermore, the X-allele carriage was associated with the decrease in lung function (forced expiratory volume at 1 s, FEV1) during follow-up in the patients with asthma (P= 0·033), the effect being strongest for non-atopic asthmatics (P= 0·042). The MBL2 genotype had no clear effect on the occurrence of atopy in adults. In conclusion, our results abrogate the previously suggested predisposing effect of MBL insufficiency on atopy at least in adults. However, as MBL is a complement component participating in immune defence against microbes, and as in the pathogenesis of non-atopic asthma infectious agents are probably involved, the gene–environment interactions between MBL and infections should be assessed further with regard to asthma.
Keywords: adult, asthma, atopy, genotype, mannose-binding lectin
Introduction
Asthma is a chronic inflammatory disorder of the lungs characterized by bronchial hyperresponsiveness, and its appearance is determined by genetic and environmental factors [1]. Immunologically, asthma is divided into two different variants: intrinsic and extrinsic, depending on the sensitization state towards environmental allergens and existence of atopy [2]. Atopy is characterized, in turn, by the presence of allergen-specific immunoglobulin E (IgE) antibodies and elevated total IgE. The skin-prick test (SPT) is usually used to examine IgE-mediated allergic responses, and the results are generally in line with anamnestic data on atopy [3].
Mannose-binding lectin (MBL) is the first component of the complement lectin pathway and an acute-phase reactant secreted by the liver [4,5]. It is encoded in humans by a single functional gene (MBL2) at chromosome 10q11.2–q21 [4–6]. Low concentration and insufficiency of MBL are caused by polymorphisms in codons 52 (CGT→TGT; designated D allele), 54 (GGC→GAC; B) and 57 (GGA→GAA; C) in exon 1 of the structural MBL2 gene, which result in amino acid substitutions Arg→Cys, Gly→Asp and Gly→Glu in the peptide, respectively [7–9]. Furthermore, MBL concentration is highly dependent upon several promoter region polymorphisms of the MBL2 gene, of which that at position −221 base pairs (bp) (nucleotide change G→C; alleles Y→X, respectively) is clinically the most important: the presence of the X-allele in the homozygous state influences serum basal MBL concentration equal to that single structural variant allele [10].
MBL insufficiency causes an opsonization defect [11–14]– mainly via decreased deposition of C3-derived opsonins [15,16] − and this has been observed to be associated with increased susceptibility to infections and atopy [17,18]. Recently, Hogaboam and colleques have observed that mannose-binding lectin insufficiency contributes to the development and maintenance of airway hyperresponsiveness during chronic fungal asthma in a mouse model [19]. Furthermore, Nagy et al. have concluded that the variant MBL2 alleles may have an important role in the susceptibility to asthma in children, especially in association with Chlamydia pneumoniae infection [20]. However, the significance of MBL insufficiency in adult persistent asthma has not been studied.
Here we studied MBL2 genotypes in adult asthmatics and controls in order to establish whether they have an effect on susceptibility to adult persistent asthma, atopy and related phenotypes.
Materials and methods
Study population
A total of 245 asthmatic patients and 405 non-asthmatic controls (mean age 60 years, range 31–89 years) participated in the study. The participants were the subjects of a Finnish population-based case–control study conducted to investigate the risk factors and predictors of the outcome of adult asthma. Detailed information on the subjects has been reported elsewhere [21]. Inclusion criteria for asthmatic subjects were age over 30 years and entitlement to reimbursement for asthma medication from the Social Insurance Institution of Finland. The diagnoses were made by chest physicians on the basis of clinical and physiological criteria described earlier [22]. Of 113 patients, 18 years (mean, range 17–20) follow-up data concerning the change in forced expiratory volume at 1 s (ΔFEV1) were available from the ‘Mini-Finland Health Survey’ study [23]. One or two controls without asthma or chronic obstructive pulmonary disease were selected initially for each subject using a registry covering the entire population. They were matched for age, sex and area of residence. Of 215 controls, ΔFEV1 follow-up data were also available.
Methods
MBL genetic polymorphism testing was carried out in 99·2% (243/245) of the asthmatic patients and 98·8% of the non-asthmatic controls (400/405). Exon 1 of the MBL2 structural gene was amplified by polymerase chain reaction (PCR) [9]. Genotyping of codon 52 (CGT→TGT; designated D or O), 54 (GGC→GAC; B or O) and 57 (GGA→GAA; C or O) polymorphisms was performed by sequencing. Structural alleles lacking these single nucleotide polymorphisms were classified as wild-type (A). For genotyping of promoter region polymorphisms at position −221 bp (G→C, designated Y- or X-alleles, respectively) commercially available fluorogenic allele-specific TaqMan probes and primers were used (rs7096206; Applied Biosystems, CA, USA). Genotyping was performed by means of the 5′ nuclease assay for allelic discrimination using the ABI Prism 7000 Sequence Detection System (Applied Biosystems).
Atopy was determined by skin-prick test performed by trained nurses with a panel of 22 allergen extracts selected to cover exposures in both urban and rural environments (ALK A/S, Copenhagen, Denmark). Detailed information on the allergen extracts has been described previously [21]. The subject was considered skin-prick test positive if at least one allergen showed a wheal with a diameter at least 3 mm larger than that of the negative control.
Statistical analysis
Proportions were compared by χ2 or Fisher's exact test and levels by Kruskall–Wallis or Mann–Whitney U-test. The results are supported by odds ratios (OR) and 95% confidence intervals (CI) when appropriate.
Ethics
The study plan was accepted by the ethical committee of Tampere University Hospital, Tampere, Finland, and all patients gave their informed consent.
Results
Distribution of MBL2 genotypes among adult patients with asthma and controls, also stratified by the occurrence of atopy, is shown in Tables 1 and 2. In the non-atopic subgroup, a clear disequilibrium was detected in promoter genotype frequencies among males (P= 0·036). In further analyses, the carriage of an X-allele proved to be a significant risk factor for intrinsic asthma in these patients (OR = 2·52, 95% CI = 1·23–5·15; P = 0·01), the proportions of X-allele carriers being 26/48 (54%) in the non-atopic male asthmatics and 30/94 (32%) in the non-atopic male controls.
Table 1. Distribution of mannose-binding lectin (MBL2) structural and promoter genotypes among 243 adult patients with asthma and 400 controls.
| Asthma | Controls | ||
|---|---|---|---|
| Genotype | n= 243 | n= 400 | P-valuea |
| Structural | |||
| A/A1,2 | 156 (64%) | 248 (62%) | |
| A/O2 | 80 (33%) | 136 (34%) | |
| A/B1 | 53 (22%) | 96 (24%) | |
| A/C1 | 4 (1·6%) | 8 (2·0%) | |
| A/D1 | 23 (9·5%) | 32 (8·0%) | |
| O/Ob1,2 | 7 (2·9%) | 16 (4·0%) | 1P = 0·8372P = 0·705 |
| Promoter | |||
| Y/Y | 157 (65%) | 264 (66%) | |
| Y/X | 72 (30%) | 116 (29%) | |
| X/X | 14 (5·8%) | 20 (5·0%) | P= 0·891 |
| Combined genotype (structural + promoter) | |||
| YA/YA | 88 (36%) | 140 (35%) | |
| YA/XA | 54 (22%) | 88 (22%) | |
| YA/YO | 62 (26%) | 108 (27%) | |
| XA/XA | 14 (5·8%) | 20 (5·0%) | |
| XA/YO | 18 (7·4%) | 28 (7·0%) | |
| YO/YO | 7 (2·9%) | 16 (4·0%) | P= 0·923 |
| MBL sufficient | |||
| (YA/YA, YA/XA or YA/YO) | 204 (84%) | 336 (84%) | |
| MBL insufficient | |||
| (XA/XA, XA/YO or YO/YO) | 39 (16%) | 64 (16%) | P= 0·987 |
χ2test;
variant with regard to both alleles: two patients with B/B, one with B/C, three with B/D and one with D/D genotype, and five controls with B/B, one with B/C, eight with B/D, one with C/D and one with D/D genotype.
Table 2. Distribution of mannose-binding lectin (MBL2) genotypes among the adult patients with asthma and the controls, stratified by the occurrence of atopy.
| Genotype, Structural | Genotype, promoter | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A/A | A/B | A/C | A/D | O/Oc | P valueb | Y/Y | Y/X | X/X | P valueb | ||
| Atopica | |||||||||||
| Asthma (n = 137) | 87 (64%) | 28 (20%) | 4 (2·9%) | 15 (11%) | 3 (2·2%) | 95 (69%) | 34 (25%) | 8 (5·8%) | |||
| Controls (n = 150) | 95 (63%) | 40 (27%) | 0 (0%) | 10 (6·7%) | 5 (3·3%) | 0·116 | 99 (66%) | 44 (29%) | 7 (4·7%) | 0·656 | |
| Non-atopic | |||||||||||
| Asthma (n = 104) | 67 (64%) | 25 (24%) | 0 (0%) | 8 (7·7%) | 4 (3·8%) | 61 (59%) | 37 (36%) | 6 (5·8%) | |||
| Controls (n = 247) | 151 (61%) | 55 (22%) | 8 (3·2%) | 22 (8·9%) | 11 (4·5%) | 0·436 | 164 (66%) | 71 (29%) | 12 (4·9%) | 0·384d | |
Data concerning atopy were not applicable from two patients with asthma and three controls. Atopy was determined by at least one positive skin-prick test;
χ2test;
variant in regard to both alleles;
significant difference in males (P= 0·036).
The effect of MBL2 promoter genotypes on the demographic and clinical characteristics of patients with persistent asthma is shown in Table 3. The association between the MBL2 promoter genotype and ΔFEV1 in follow-up did not reach the statistically significant level (P= 0·059). However, in X-allele carriers ΔFEV1 decreased 55 ml/year (median, quartiles48–76 ml/year),whileinthosepatientswiththeY/Y genotype the decrease was only 49 ml/year (median, quartiles 35–66 ml/year) (P= 0·033). This phenomenon could not be detected in controls (n = 215; P = 0·147). In further subgroup analyses the X-allele effect was significant, especially in patients with non-atopic asthma (P= 0·042, the X-allele effect in atopic asthma P = 0·756). The MBL2 structural genotype had no effect on the characteristics shown in Table 3.
Table 3. The effect of mannose-binding lectin (MBL2) promoter genotypes on the demographic and clinical characteristics of the patients with persistent asthma. MBL2 structural genotypes had no effect on these characteristics. The values are expressed as proportions or medians (quartiles), if not otherwise stated.
| Genotype, promoter | ||||
|---|---|---|---|---|
| Character | Y/Y | Y/X | X/X | P-valuec |
| Sex, female | 99/157 (63%) | 44/72 (61%) | 8/14 (57%) | 0·888 |
| Age at diagnosis, year | 48 (40–57) | 49 (38–58) | 46 (37–55) | 0·750 |
| Atopy | 95/156 (61%) | 34/71 (48%) | 8/14 (57%) | 0·186 |
| B-eosinophil cell count, × 109/l | 0·14 (0·09–0·27) | 0·19 (0·12–0·29) | 0·13 (0·05–0·25) | 0·120 |
| S-IgE, kU/l | 54 (15–200) | 38 (15–103) | 20 (10–106) | 0·148 |
| aFEV1 predicted, % | 89 (71–106) | 86 (70–101) | 94 (80–108) | 0·353 |
| bΔFEV1, ml/ year, n = 113 | 49 (35–66) | 55 (47–72) | 61 (55–89) | 0·059 |
Predicted percentage of forced expiratory volume at 1 s;
change in FEV1 in follow-up;
χ2 or Kruskall—Wallis test.
The association between MBL2 genotype and atopy in the controls, stratified by gender, is shown in Table 4. No statistically significant findings were detected. However, when evaluating the clinical symptoms in the controls with atopy, lower respiratory tract symptoms seemed to be less frequent in females with the heterozygous variant A/O genotype compared to those females with the wild-type A/A genotype [3/31 (9·7%) and 19/61 (31%), respectively; P = 0·037].
Table 4. The association between mannose-binding lectin (MBL2) genotypes and atopy in controls, stratified by gender.
| Genotype, structural | Genotype, promoter | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Controlsa | A/A | A/B | A/C | A/D | O/Oc | P-valueb | Y/Y | Y/X | X/X | P-valueb |
| All (n = 397), atopic (%) | 95 (39%) | 40 (42%) | 0 (0%) | 10 (31%) | 5 (31%) | 0·161 | 99 (38%) | 44 (38%) | 7 (37%) | 0·990 |
| Males (n = 150), atopic (%) | 34 (38%) | 12 (34%) | 0 (0%) | 7 (47%) | 3 (38%) | 0·755 | 39 (38%) | 16 (39%) | 1 (17%) | 0·560 |
| Females (n = 247), atopic (%) | 61 (39%) | 28 (47%) | 0 (0%) | 3 (18%) | 2 (25%) | 0·056 | 60 (38%) | 28 (38%) | 6 (46%) | 0·825 |
Data concerning atopy were not applicable from three controls. Atopy was determined by at least one positive skin-prick test;
χ2 test;
variant with regard to both alleles.
Discussion
We investigated the significance of MBL2 variant genotypes causing low MBL concentration and MBL insufficiency on the occurrence of adult persistent asthma and atopy. The population-based study material consisted of 243 adult patients with persistent asthma and 400 controls. Among the latter, the proportions of the structural variant genotypes were similar to those described previously in Finnish and other Caucasian populations, the B allele being the most common variant form [4,24]. The proportions of the promoter variant genotypes have not been studied previously in Finns. However, they seemed to be similar to those reported in other Caucasians [10].
MBL insufficiency has been observed previously to contribute to the development and maintenance of airway hyperresponsiveness during chronic fungal asthma in a mouse model, but it does not participate in chronic airway remodelling [19]. Furthermore, MBL2 variant alleles have been detected to influence susceptibility to asthma in children only in conjunction with C. pneumoniae infection [20]. In this study we showed that the carriage of a promoter region variant allele X causing low MBL expression is a significant risk factor for asthma in non-atopic males. Furthermore, the X-allele was associated with the decrease in FEV1 during follow-up in patients with asthma, the effect being strongest for non-atopic asthmatics. It is known that the aetiopathogenesis of atopic and non-atopic asthma differs and the pathogenesis of non-atopic asthma is suggested to be related to infections: for example, the association between elevated C. pneumoniae antibodies and asthma is strongest for the non-atopic disease form [25]. Furthermore, persistent airflow limitation in adult-onset non-atopic asthma is associated with serological evidence of C. pneumoniae infection [26]. MBL insufficiency is highly associated with increased susceptibility to recurrent respiratory infections both in adults and children [27–29]. It is possible that MBL insufficiency due to promoter region polymorphism leads to impaired recovery from lower respiratory tract infections, causes prolonged immunological hyperreactivity in the airways and plays a role in the pathogenesis of intrinsic asthma.
The predisposing effect of the X-allele on intrinsic asthma was seen only in males. It is known that there are gender differences in immune mechanisms and in responses against invading microbes, and after puberty females run a higher risk of developing new asthma [30–32]. Furthermore, the predisposing effect of the MBL2 genotype was restricted only to the promoter region X-allele instead of the structural variants. Structural and promoter genotypes manifest differently: the structural variants distort the formation and structure of the MBL molecule, while the X-allele influences negatively the expression of normally functioning MBL, which may be a significant factor in the early state of an innate immune response against, e.g. microbes [4,5,10].
When evaluating the effect of MBL2 genotype on the occurrence of atopy in controls, no statistically significant findings were detected. However, when evaluating the clinical symptoms among the controls with atopy, lower respiratory tract symptoms tend to be less frequent in females carrying the A/O genotype than in those females with the A/A genotype. Differences in gender distribution concerning atopy and related disorders have been detected previously [32]. The opsonization defect caused by MBL insufficiency has been observed to be associated with atopy in children [17,18]. However, the direct connection between MBL insufficiency and atopy has not been studied previously. Our results overturn the previously suggested predisposing effect of MBL insufficiency on atopy, at least in adults. In fact, MBL insufficiency may even provide protection against atopic symptoms in certain subgroups.
In conclusion, the MBL2 genotype had no predisposing effect on the occurrence of atopy; thus, our results abrogate the previously suggested risk effect of MBL insufficiency on atopy at least in adults. However, we have show that the carriage of a promoter region variant allele X causing low MBL expression is a significant risk factor for asthma in non-atopic males. Furthermore, the X-allele carriage was associated with the decrease in FEV1 during follow-up in patients with asthma, the effect being strongest for non-atopic asthmatics. Because no significant association was seen with MBL2 structural genotypes, the results should be interpreted with caution. However, as MBL is a complement component participating in immune defence against microbes, and as in the pathogenesis of non-atopic asthma infectious agents are involved, the gene–environment interactions between MBL and infections should be assessed further with regard to asthma.
Acknowledgments
This study was supported financially by the Rehabilitation Funds of the Finnish Social Insurance Institution, the Medical Research Fund of Tampere University Hospital, and the Tampere Tuberculosis Foundation. We thank Doctors Timo Klaukka and Arpo Aromaa for their part in collecting the research material and Marjo Leponiemi for her expert technical assistance.
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