The CD14 C-159T polymorphism is not associated with asthma or asthma severity in an Australian adult population
http://www.100md.com
《胸》
1 Asthma and Allergy Research Institute Inc and Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, Perth, Australia
2 Cooperative Research Centre for Asthma, University of Western Australia, Perth, Autralia
3 Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Perth, Australia
4 Genetic Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia
5 Telethon Institute for Child Health Research, Perth, Australia
Correspondence to:
Associate Professor P J Thompson
Asthma and Allergy Research Institute, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia; aari@aari.uwa.edu.au
Received 18 May 2004
Revised version received 19 December 2004
ABSTRACT
Background: CD14 functions as a multifunctional receptor for bacterial cell wall components including endotoxin and lipopolysaccharide and is likely to play a role in the polarisation of T lymphocytes into Th1 and Th2 subsets, thereby influencing the cytokine profile and subsequent IgE production in response to antigen/allergen contact in allergic phenotypes. A functional C-159T polymorphism has been described in the promoter region of the gene and has been associated with increased gene expression, atopy, and non-atopic asthma in different ethnic populations. A study was undertaken to examine the association between the C-159T polymorphism and asthma, asthma severity, and atopy in a large Australian white population.
Methods: PCR-RFLP analysis was used to characterise the C-159T polymorphism in mild (n = 264), moderate (n = 225) and severe (n = 79) asthmatic patients and non-asthmatic controls (n = 443), including atopic (n = 688) and non-atopic (n = 323) individuals. Association analyses were performed using 2 tests.
Results: There was no association between the polymorphism and asthma (p = 0.468) or asthma severity (p = 0.727), and only a very weak association with atopy (p = 0.084). A meta-analysis of all studies conducted to date revealed similar genotypic frequencies in white ethnic populations and confirmed that there was no overall association with atopy (p = 0.52) or asthma (p = 0.23), although there was significant between study heterogeneity (p = 0.01).
Conclusions: This study confirms that there is no association between the CD14 C-159T polymorphism and asthma or asthma severity and a weak association between this polymorphism and atopy in an adult population.
Keywords: CD14; genetics; asthma; atopy
CD14 is a multifunctional receptor which is constitutively expressed on the surface of monocytes, macrophages, and neutrophils and as a soluble form in serum.1 It is the principal receptor for lipopolysaccharide (LPS) or inhaled endotoxin, a potent inducer of lung inflammation which may activate innate immune pathways that promote Th1 differentiation and/or suppress Th2 dependent immunoglobulin E (IgE) responses. CD14 binding of LPS is associated with a strong IL-12 response by antigen presenting cells,2,3 and IL-12 is regarded as an obligatory signal for the maturation of na?ve T cells into Th1 cells.3 IgE responses are regulated by inhibitory signals derived from Th1 type cells and by stimulatory signals from Th2 type cells.4 It is proposed that altered CD14 expression may affect the proportion of Th1 to Th2 cells, thereby influencing IgE responses5 and the associated inflammatory phenotype in allergic conditions such as asthma.
It has been suggested that asthmatic subjects are more sensitive to the effects of LPS than non-asthmatic subjects,6 and subjects with allergic asthma have been reported to have increased expression of CD14 after acute allergen challenge7 and LPS inhalation.8 Thus, alterations in CD14 expression appear to be important, particularly in allergic asthma, and it is likely that its expression is regulated, at least partially, at the gene level. A functional single nucleotide polymorphism (C-159T) has been described in the promoter region of the CD14 gene5 and has been associated with altered CD14 and IgE levels in various ethnic populations.5,9–12
Previous studies have investigated the promoter polymorphism in populations of varying sizes and ethnicity, with limited definitions of asthma severity. We sought to investigate the association between the C-159T polymorphism and atopy and to determine if there was an association between this polymorphism and asthma and/or asthma severity in a large, carefully phenotyped adult Australian white ethnic population of non-asthmatic controls and patients with mild, moderate, and severe asthma.
METHODS
Subjects
Four hundred and forty three control individuals with no evidence of asthma, 264 individuals with mild asthma, 225 with moderate asthma, and 79 with severe asthma participated in this association study. The population, methods of recruitment, and disease severity categorisation have been described previously.13 All subjects were unrelated and completed a detailed questionnaire and spirometric tests, and blood samples were obtained for DNA extraction. All individuals were tested for atopic status by skin prick testing, with a positive reaction resulting in a wheal of more than 3 mm diameter to at least one of the five aeroallergens tested (including cat, dog, house dust mite, mould mix and grass pollen mix).
The study protocol was approved by the Sir Charles Gairdner Hospital human research ethics committee and all subjects provided informed written consent to participate in this study.
Molecular methods
DNA was extracted from buffy coats using a commercially available DNA extraction kit (Qiagen, Hilden, Germany), following the manufacturers’ instructions. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis was optimised following the protocol described by Baldini et al.5 At final volume the PCR reaction contained 1x PCR buffer (Qiagen), 1x Q solution (Qiagen), 2 mM MgCl2, 200 μM each of dATP, dTTP, dCTP and dGTP (Promega, Madison, USA), 20 pmol of each primer (5'-GTGCCAACAGATGAGGTTCAC-3' and 5'-GCCTCTGACAGTTTTATGTAATC-3'), and 1 U of Taq DNA polymerase (Qiagen). PCR amplification conditions were optimised with an initial denaturation step at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute, and a final extension step at 72°C for 10 minutes in a thermal cycler (Eppendorf, Hamburg, Germany).
The restriction endonuclease AvaII recognises the restriction sequence GGTCC, present only in amplified DNA fragments containing the T allele. The restriction mix contained 1x restriction buffer C (Promega, Madison, WI, USA) and 1 U AvaII (Promega). Restricted products were electrophoresed on 1% agarose gels (Amresco, OH, USA). Expected fragment sizes were 497 bp for the C allele and 353+144 bp for the T allele.
Statistical methods
Genotype and allele frequencies were calculated for each phenotypic group. Comparisons of allele and genotype distribution were performed with 2 tests using the R Statistics Program.14 The influence of the CD14 promoter polymorphism on specific asthma phenotypes was also examined using multivariate logistic regression analysis, and Hardy-Weinberg equilibrium analysis for each group was evaluated by the exact test implemented in the R Statistics Program. We performed a meta-analysis using the tabulated results from several populations: American white (atopic n = 161, non-atopic n = 122;5 atopic asthma n = 99, non-atopic asthma n = 43, controls n = 4915); Polish (atopic asthma n = 50, controls n = 7316); Japanese (atopic asthma n = 100, non-atopic asthma n = 100, controls n = 10017); German (asthma n = 84, atopic n = 78, non-atopic n = 77, controls n = 119;18 asthma n = 182, non-asthma n = 26119); British (atopic asthma n = 125, controls n = 15017); Dutch (atopic n = 172, non-atopic n = 14312); Chinese (asthma n = 258, non-asthma n = 92, atopic n = 261, non-atopic n = 8920); Czechoslovakian (atopic n = 37, non-atopic n = 10111); Icelandic (atopic asthma n = 94, controls n = 9421); and Australian (asthma n = 46322). For some studies, where genotypic frequencies were not available, we estimated genotypic counts based on the allele frequencies assuming Hardy-Weinberg equilibrium. The meta-analysis was performed using both fixed and random effects binomial regression, the latter using the approach of Whitehead and Whitehead.23
RESULTS
The CD14 C-159T promoter polymorphism was characterised in a large carefully phenotyped population of 1011 unrelated Australian white ethnic subjects described previously:13 all subgroups had similar age and sex distributions and the asthma group as a whole had a significantly higher percentage of atopic subjects (83%) than the non-asthmatic control group (49%; p<0.0001).
Table 1 summarises the genotype analysis for all phenotypic groups. All groups studied were in Hardy-Weinberg equilibrium (p0.402). There was no association between the C-159T polymorphism and asthma (p = 0.468) or disease severity (p = 0.727) in this population, although the T allele was slightly more common in patients with asthma, particularly those with mild and severe asthma. This effect was not influenced by age or sex. There was a very weak association between the C-159T polymorphism and atopy (p = 0.084), with the T allele slightly less common in non-atopic individuals.
Table 1 CD14 C-159T genotype and allele frequencies in an Australian adult population
We performed a meta-analysis using the tabulated results from several populations and, in a first analysis comparing healthy controls, all populations were shown to have similar genotypic frequencies with the exception of a Japanese population.9 Both random effects and fixed effects logistic regression models concluded no overall association between asthma and CD14 genotype (p = 0.23, pooled allelic odds ratio 1.16, 95% CI 0.98 to 1.39). For atopy, again there was no overall association with the CD14 genotype (p = 0.52, pooled allelic odds ratio 0.83, 95% CI 0.58 to 1.20) but there was significant between-study heterogeneity (p = 0.01).
DISCUSSION
A functional C-159T polymorphism has been described in the promoter region of the CD14 gene and has been associated with increased CD14 expression in vitro24 and in the serum of children,5,20 and with altered serum IgE levels and skin test positivity in different populations.5,9–12,20,25,26 We have confirmed a weak association between the C-159T polymorphism and atopy but found no association between this polymorphism and asthma or asthma severity in a large well phenotyped Australian white adult population.
The CD14 gene has been localised to chromosome 5q31, in a region shown to be linked to Th2 prevalent phenotypes including high total serum IgE levels and asthma, in a number of different populations.27–35 Several polymorphisms have been described and investigated in the CD14 gene5,11,19,26,36 and, of these, the C-159T promoter polymorphism has been the most extensively studied in atopic disease. In vitro studies using transient transfection assays in CD14 expressing monocytic cells showed that the C-159T polymorphism increases transcription by lowering the affinity of the CD14 regulatory region for Sp3,24 a factor known to inhibit the activity of a number of promoters.37–41 In clinical studies the C-159T TT genotype has been associated with higher circulating sCD14, a lower mean number of positive skin tests, and lower serum IgE levels in British and American white populations,5,9,26 and has also been found to be associated with non-atopic asthma and food allergy in various ethnic groups.15 Conversely, the CC genotype has been associated with higher levels of total serum IgE and a higher number of positive skin tests in a population from the Netherlands12,20,26 and the C allele has been associated with atopy, specifically to moulds, in a Czechoslovakian population.11
However, the associations between the C-159T polymorphism and atopic phenotypes have not always been consistent and, in contrast to these other findings, the CD14-159T allele was over-transmitted to atopic individuals in an inbred Hutterite population. Similarly, we have shown that the -159T allele was slightly more common in atopic adults. There was no association of this polymorphism with atopy in an Hispanic population,5 with atopic asthma in an Icelandic population,21 or with atopy and/or asthma in two German white populations.18,19
Although some populations have been relatively small, the allele frequencies of the C-159T polymorphism have not varied significantly between all populations investigated to date, with the exception of a Japanese population.9 The -159T allele frequencies ranged between 0.60 in a Japanese population,9 0.53 in a Chinese population,20 and 0.37 and 0.54 in all white populations: 0.37–0.41 in American;5,15 0.38 in Polish;16 0.42–0.46 in German;18,19 0.45 in Icelandic;21 0.47 in Australian; 0.52 in British;9 0.53 in Dutch;12 0.54 in Czechoslovakian.11 A meta-analysis of all populations studied to date revealed that there was no overall association between the C-159T polymorphism and asthma (p = 0.23) or atopy (p = 0.52); however, it did reveal significant between-study heterogeneity (p = 0.01).
It is difficult to explain the inconsistency of association studies for the C-159T polymorphism, even between populations of similar ethnicity. Environmental factors may differ between the study populations18 but, as has been highlighted by a number of authors, association studies are commonly difficult to replicate between different populations, especially when different phenotypic markers are analysed.18,19
There is tight linkage disequilibrium across the CD14 promoter region42 which would strongly favour linkage between C-159T and polymorphisms in CD14 or another gene on 5q, and may account for some of the associations seen to date. In support of this, the CD14-159T allele has been shown to be over-transmitted to atopic individuals in an inbred Hutterite population, only when on a haplotype with marker D5S642 previously shown to be linked to atopy in this population.43 It is therefore highly likely that the CD14-159T allele is actually in linkage disequilibrium with the susceptibility variant on 5q.10
It is also possible that the C-159T polymorphism has an age related influence on the development of atopy. The polymorphism has been associated with increased CD14 expression only in the serum of children,5,20 and there was no difference in serum CD14 levels or the expression of membrane bound levels of CD14 in a sample of adult blood donors with different CD14 genotypes.44 Additionally, a longitudinal study in Australian white subjects aged 8–25 years found that individuals with the CC genotype were more likely to have early onset atopy and early onset airway hyperresponsiveness, suggesting that the influence of -159C on the atopic genotype may be age specific.25 There is strong evidence to suggest that atopy and asthma are closely related entities, with a positive association between total serum IgE and number of positive skin tests, bronchial hyperresponsiveness, and development of asthma and doctor diagnosed asthma.45,46 Thus, alleles which are associated with atopy might be expected to be over-represented in individuals with asthma relative to the general population.12 However, to date, no association has been found between the C-159T polymorphism and atopic asthma. We have confirmed this in our population and in a meta-analysis of all populations studied, suggesting that separate genes on chromosome 5q may regulate susceptibility to high total serum IgE levels and bronchial responsiveness.5
Similarly, there are no publications showing an association between the CD14 C-159T polymorphism and asthma severity, although it has been suggested that it might modify the severity of airflow obstruction in asthmatics. A recent conference abstract reported a study conducted on 418 Australian adult asthmatics22 in which neither the CT nor TT genotypes were associated with life threatening asthma, although the TT genotype was found to be weakly associated with lower forced expiratory volume in 1 second (FEV1). However, there was no association between genotype and asthma severity (also defined by mean FEV1) in a smaller predominantly white American population.15 We have used a more extensive set of criteria to define asthma severity13 and have confirmed that there is no relationship between asthma severity and the C-159T polymorphism in our population.
ACKNOWLEDGEMENTS
The authors thank the patients for their participation in this study and the staff at Sir Charles Gairdner Hospital and the Asthma and Allergy Research Institute for all their assistance with patient recruitment.
FOOTNOTES
This project was funded by the Asthma and Allergy Research Institute, the Asthma Foundation of Western Australia, the NH&MRC and the Australian Medical Students Association. David Duffy is an NH&MRC Research Fellow.
REFERENCES
Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 1995;13:437–57.
Cleveland MG, Gorham JD, Murphy TL, et al. Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway. Infect Immun 1996;64:1906–12.
Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+T cells. J Immunol 1995;154:5071–9.
Romagnani S. Induction of TH1 and TH2 responses: a key role for the ‘natural’ immune response? Immunol Today 1992;13:379–81.
Baldini M, Lohman IC, Halonen M, et al. A polymorphism in the 5' flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am J Respir Cell Mol Biol 1999;20:976–83.
Peden DB, Tucker K, Murphy P, et al. Eosinophil influx to the nasal airway after local, low-level LPS challenge in humans. J Allergy Clin Immunol 1999;104:388–94.
Virchow JC Jr, Julius P, Matthys H, et al. CD14 expression and soluble CD14 after segmental allergen provocation in atopic asthma. Eur Respir J 1998;11:317–23.
Alexis N, Eldridge M, Reed W, et al. CD14-dependent airway neutrophil response to inhaled LPS: role of atopy. J Allergy Clin Immunol 2001;107:31–5.
Gao PS, Mao XQ, Baldini M, et al. Serum total IgE levels and CD14 on chromosome 5q31. Clin Genet 1999;56:164–5.
Ober C, Tsalenko A, Parry R, et al. A second-generation genomewide screen for asthma-susceptibility alleles in a founder population. Am J Hum Genet 2000;67:1154–62.
Buckova D, Holla LI, Schuller M, et al. Two CD14 promoter polymorphisms and atopic phenotypes in Czech patients with IgE-mediated allergy. Allergy 2003;58:1023–6.
Koppelman GH, Reijmerink NE, Stine CO, et al. Association of a promoter polymorphism of the CD14 gene and atopy. Am J Respir Crit Care Med 2001;163:965–9.
Kedda MA, Shi J, Duffy DL, et al. Characterisation of two polymorphisms in the LTC4 synthase gene in an Australian population of mild, moderate and severe asthmatics. J Allergy Clin Immunol 2004;113:889–95.
R Development Core Team. R: A language and environment for statistical computing. Vienna, Austria, 2003 ( http://www.R-project.org ).
Woo JG, Assa’ad A, Heizer AB, et al. The -159 C–>T polymorphism of CD14 is associated with nonatopic asthma and food allergy. J Allergy Clin Immunol 2003;112:438–44.
Lis G, Kostyk E, Sanak M, et al. . Pneumonol Alergol Pol 2001;69:265–72.
Gao PS, Mao XQ, Kawai M, et al. Negative association between asthma and variants of CC16(CC10) on chromosome 11q13 in British and Japanese populations. Hum Genet 1998;103:57–9.
Sengler C, Haider A, Sommerfeld C, et al. Evaluation of the CD14 C-159 T polymorphism in the German Multicenter Allergy Study cohort. Clin Exp Allergy 2003;33:166–9.
Heinzmann A, Dietrich H, Jerkic SP, et al. Promoter polymorphisms of the CD14 gene are not associated with bronchial asthma in Caucasian children. Eur J Immunogenet 2003;30:345–8.
Leung TF, Tang NL, Sung YM, et al. The C-159T polymorphism in the CD14 promoter is associated with serum total IgE concentration in atopic Chinese children. Pediatr Allergy Immunol 2003;14:255–60.
Hakonarson H, Bjornsdottir US, Ostermann E, et al. Allelic frequencies and patterns of single-nucleotide polymorphisms in candidate genes for asthma and atopy in Iceland. Am J Respir Crit Care Med 2001;164:2036–44.
Savirimuthu S, Yang IA, Shaw J, et al. CD14 polymorphisms and disease severity in asthmatics. Thoracic Society of Australasia and New Zealand. Adelaide, Australia. Respirology 2003;S2:A27.
Whitehead A, Whitehead J. A general parametric approach to the meta-analysis of randomized clinical trials. Stat Med 1991;10:1665–77.
LeVan TD, Bloom JW, Bailey TJ, et al. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity. J Immunol 2001;167:5838–44.
O’Donnell AR, Toelle BG, Marks GB, et al. Age-specific relationship between CD14 and atopy in a cohort assessed from age eight to twenty-five. Am J Respir Crit Care Med 2003;169:615–22.
Vercelli D, Baldini M, Stern D, et al. CD14: a bridge between innate immunity and adaptive IgE responses. J Endotoxin Res 2001;7:45–8.
Marsh DG, Neely JD, Breazeale DR, et al. Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 1994;264:1152–6.
Meyers DA, Postma DS, Panhuysen CI, et al. Evidence for a locus regulating total serum IgE levels mapping to chromosome 5. Genomics 1994;23:464–70.
Daniels SE, Bhattacharrya S, James A, et al. A genome-wide search for quantitative trait loci underlying asthma. Nature 1996;383:247–50.
Doull IJ, Lawrence S, Watson M, et al. Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness. Am J Respir Crit Care Med 1996;153:1280–4.
(CSGA ) Collaborative Study on the Genetics of Asthma. A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet 1997;15:389–92.
Noguchi E, Shibasaki M, Arinami T, et al. Evidence for linkage between asthma/atopy in childhood and chromosome 5q31–q33 in a Japanese population. Am J Respir Crit Care Med 1997;156:1390–3.
Hizawa N, Freidhoff LR, Ehrlich E, et al. Genetic influences of chromosomes 5q31–q33 and 11q13 on specific IgE responsiveness to common inhaled allergens among African American families. Collaborative Study on the Genetics of Asthma (CSGA). J Allergy Clin Immunol 1998;102:449–53.
Martinez FD, Solomon S, Holberg CJ, et al. Linkage of circulating eosinophils to markers on chromosome 5q. Am J Respir Crit Care Med 1998;158:1739–44.
Palmer LJ, Daniels SE, Rye PJ, et al. Linkage of chromosome 5q and 11q gene markers to asthma-associated quantitative traits in Australian children. Am J Respir Crit Care Med 1998;158:1825–30.
Lazarus R, Vercelli D, Palmer LJ, et al. Single nucleotide polymorphisms in innate immunity genes: abundant variation and potential role in complex human disease. Immunol Rev 2002;190:9–25.
Hagen G, Muller S, Beato M, et al. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 1994;13:3843–51.
Hata Y, Duh E, Zhang K, et al. Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 1998;273:19294–303.
Majello B, De Luca P, Lania L. Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem 1997;272:4021–6.
Birnbaum MJ, van Wijnen AJ, Odgren PR, et al. Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3. Biochemistry 1995;34:16503–8.
Dennig J, Hagen G, Beato M, et al. Members of the Sp transcription factor family control transcription from the uteroglobin promoter. J Biol Chem 1995;270:12737–44.
Vercelli D, Baldini M, Martinez F. The monocyte/IgE connection: may polymorphisms in the CD14 gene teach us about IgE regulation? Int Arch Allergy Immunol 2001;124:20–4.
Ober C, Tsalenko A, Willadsen S, et al. Genome-wide screen for atopy susceptibility alleles in the Hutterites. Clin Exp Allergy 1999;29 (Suppl 4) :11–5.
Heesen M, Blomeke B, Schluter B, et al. Lack of association between the -260 C–>T promoter polymorphism of the endotoxin receptor CD14 gene and the CD14 density of unstimulated human monocytes and soluble CD14 plasma levels. Intensive Care Med 2001;27:1770–5.
Sears MR, Herbison GP, Holdaway MD, et al. The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma. Clin Exp Allergy 1989;19:419–24.
Burrows B, Martinez FD, Halonen M, et al. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989;320:271–7.(M-A Kedda1,2,3, F Lose1,3)
2 Cooperative Research Centre for Asthma, University of Western Australia, Perth, Autralia
3 Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Perth, Australia
4 Genetic Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia
5 Telethon Institute for Child Health Research, Perth, Australia
Correspondence to:
Associate Professor P J Thompson
Asthma and Allergy Research Institute, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia; aari@aari.uwa.edu.au
Received 18 May 2004
Revised version received 19 December 2004
ABSTRACT
Background: CD14 functions as a multifunctional receptor for bacterial cell wall components including endotoxin and lipopolysaccharide and is likely to play a role in the polarisation of T lymphocytes into Th1 and Th2 subsets, thereby influencing the cytokine profile and subsequent IgE production in response to antigen/allergen contact in allergic phenotypes. A functional C-159T polymorphism has been described in the promoter region of the gene and has been associated with increased gene expression, atopy, and non-atopic asthma in different ethnic populations. A study was undertaken to examine the association between the C-159T polymorphism and asthma, asthma severity, and atopy in a large Australian white population.
Methods: PCR-RFLP analysis was used to characterise the C-159T polymorphism in mild (n = 264), moderate (n = 225) and severe (n = 79) asthmatic patients and non-asthmatic controls (n = 443), including atopic (n = 688) and non-atopic (n = 323) individuals. Association analyses were performed using 2 tests.
Results: There was no association between the polymorphism and asthma (p = 0.468) or asthma severity (p = 0.727), and only a very weak association with atopy (p = 0.084). A meta-analysis of all studies conducted to date revealed similar genotypic frequencies in white ethnic populations and confirmed that there was no overall association with atopy (p = 0.52) or asthma (p = 0.23), although there was significant between study heterogeneity (p = 0.01).
Conclusions: This study confirms that there is no association between the CD14 C-159T polymorphism and asthma or asthma severity and a weak association between this polymorphism and atopy in an adult population.
Keywords: CD14; genetics; asthma; atopy
CD14 is a multifunctional receptor which is constitutively expressed on the surface of monocytes, macrophages, and neutrophils and as a soluble form in serum.1 It is the principal receptor for lipopolysaccharide (LPS) or inhaled endotoxin, a potent inducer of lung inflammation which may activate innate immune pathways that promote Th1 differentiation and/or suppress Th2 dependent immunoglobulin E (IgE) responses. CD14 binding of LPS is associated with a strong IL-12 response by antigen presenting cells,2,3 and IL-12 is regarded as an obligatory signal for the maturation of na?ve T cells into Th1 cells.3 IgE responses are regulated by inhibitory signals derived from Th1 type cells and by stimulatory signals from Th2 type cells.4 It is proposed that altered CD14 expression may affect the proportion of Th1 to Th2 cells, thereby influencing IgE responses5 and the associated inflammatory phenotype in allergic conditions such as asthma.
It has been suggested that asthmatic subjects are more sensitive to the effects of LPS than non-asthmatic subjects,6 and subjects with allergic asthma have been reported to have increased expression of CD14 after acute allergen challenge7 and LPS inhalation.8 Thus, alterations in CD14 expression appear to be important, particularly in allergic asthma, and it is likely that its expression is regulated, at least partially, at the gene level. A functional single nucleotide polymorphism (C-159T) has been described in the promoter region of the CD14 gene5 and has been associated with altered CD14 and IgE levels in various ethnic populations.5,9–12
Previous studies have investigated the promoter polymorphism in populations of varying sizes and ethnicity, with limited definitions of asthma severity. We sought to investigate the association between the C-159T polymorphism and atopy and to determine if there was an association between this polymorphism and asthma and/or asthma severity in a large, carefully phenotyped adult Australian white ethnic population of non-asthmatic controls and patients with mild, moderate, and severe asthma.
METHODS
Subjects
Four hundred and forty three control individuals with no evidence of asthma, 264 individuals with mild asthma, 225 with moderate asthma, and 79 with severe asthma participated in this association study. The population, methods of recruitment, and disease severity categorisation have been described previously.13 All subjects were unrelated and completed a detailed questionnaire and spirometric tests, and blood samples were obtained for DNA extraction. All individuals were tested for atopic status by skin prick testing, with a positive reaction resulting in a wheal of more than 3 mm diameter to at least one of the five aeroallergens tested (including cat, dog, house dust mite, mould mix and grass pollen mix).
The study protocol was approved by the Sir Charles Gairdner Hospital human research ethics committee and all subjects provided informed written consent to participate in this study.
Molecular methods
DNA was extracted from buffy coats using a commercially available DNA extraction kit (Qiagen, Hilden, Germany), following the manufacturers’ instructions. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis was optimised following the protocol described by Baldini et al.5 At final volume the PCR reaction contained 1x PCR buffer (Qiagen), 1x Q solution (Qiagen), 2 mM MgCl2, 200 μM each of dATP, dTTP, dCTP and dGTP (Promega, Madison, USA), 20 pmol of each primer (5'-GTGCCAACAGATGAGGTTCAC-3' and 5'-GCCTCTGACAGTTTTATGTAATC-3'), and 1 U of Taq DNA polymerase (Qiagen). PCR amplification conditions were optimised with an initial denaturation step at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute, and a final extension step at 72°C for 10 minutes in a thermal cycler (Eppendorf, Hamburg, Germany).
The restriction endonuclease AvaII recognises the restriction sequence GGTCC, present only in amplified DNA fragments containing the T allele. The restriction mix contained 1x restriction buffer C (Promega, Madison, WI, USA) and 1 U AvaII (Promega). Restricted products were electrophoresed on 1% agarose gels (Amresco, OH, USA). Expected fragment sizes were 497 bp for the C allele and 353+144 bp for the T allele.
Statistical methods
Genotype and allele frequencies were calculated for each phenotypic group. Comparisons of allele and genotype distribution were performed with 2 tests using the R Statistics Program.14 The influence of the CD14 promoter polymorphism on specific asthma phenotypes was also examined using multivariate logistic regression analysis, and Hardy-Weinberg equilibrium analysis for each group was evaluated by the exact test implemented in the R Statistics Program. We performed a meta-analysis using the tabulated results from several populations: American white (atopic n = 161, non-atopic n = 122;5 atopic asthma n = 99, non-atopic asthma n = 43, controls n = 4915); Polish (atopic asthma n = 50, controls n = 7316); Japanese (atopic asthma n = 100, non-atopic asthma n = 100, controls n = 10017); German (asthma n = 84, atopic n = 78, non-atopic n = 77, controls n = 119;18 asthma n = 182, non-asthma n = 26119); British (atopic asthma n = 125, controls n = 15017); Dutch (atopic n = 172, non-atopic n = 14312); Chinese (asthma n = 258, non-asthma n = 92, atopic n = 261, non-atopic n = 8920); Czechoslovakian (atopic n = 37, non-atopic n = 10111); Icelandic (atopic asthma n = 94, controls n = 9421); and Australian (asthma n = 46322). For some studies, where genotypic frequencies were not available, we estimated genotypic counts based on the allele frequencies assuming Hardy-Weinberg equilibrium. The meta-analysis was performed using both fixed and random effects binomial regression, the latter using the approach of Whitehead and Whitehead.23
RESULTS
The CD14 C-159T promoter polymorphism was characterised in a large carefully phenotyped population of 1011 unrelated Australian white ethnic subjects described previously:13 all subgroups had similar age and sex distributions and the asthma group as a whole had a significantly higher percentage of atopic subjects (83%) than the non-asthmatic control group (49%; p<0.0001).
Table 1 summarises the genotype analysis for all phenotypic groups. All groups studied were in Hardy-Weinberg equilibrium (p0.402). There was no association between the C-159T polymorphism and asthma (p = 0.468) or disease severity (p = 0.727) in this population, although the T allele was slightly more common in patients with asthma, particularly those with mild and severe asthma. This effect was not influenced by age or sex. There was a very weak association between the C-159T polymorphism and atopy (p = 0.084), with the T allele slightly less common in non-atopic individuals.
Table 1 CD14 C-159T genotype and allele frequencies in an Australian adult population
We performed a meta-analysis using the tabulated results from several populations and, in a first analysis comparing healthy controls, all populations were shown to have similar genotypic frequencies with the exception of a Japanese population.9 Both random effects and fixed effects logistic regression models concluded no overall association between asthma and CD14 genotype (p = 0.23, pooled allelic odds ratio 1.16, 95% CI 0.98 to 1.39). For atopy, again there was no overall association with the CD14 genotype (p = 0.52, pooled allelic odds ratio 0.83, 95% CI 0.58 to 1.20) but there was significant between-study heterogeneity (p = 0.01).
DISCUSSION
A functional C-159T polymorphism has been described in the promoter region of the CD14 gene and has been associated with increased CD14 expression in vitro24 and in the serum of children,5,20 and with altered serum IgE levels and skin test positivity in different populations.5,9–12,20,25,26 We have confirmed a weak association between the C-159T polymorphism and atopy but found no association between this polymorphism and asthma or asthma severity in a large well phenotyped Australian white adult population.
The CD14 gene has been localised to chromosome 5q31, in a region shown to be linked to Th2 prevalent phenotypes including high total serum IgE levels and asthma, in a number of different populations.27–35 Several polymorphisms have been described and investigated in the CD14 gene5,11,19,26,36 and, of these, the C-159T promoter polymorphism has been the most extensively studied in atopic disease. In vitro studies using transient transfection assays in CD14 expressing monocytic cells showed that the C-159T polymorphism increases transcription by lowering the affinity of the CD14 regulatory region for Sp3,24 a factor known to inhibit the activity of a number of promoters.37–41 In clinical studies the C-159T TT genotype has been associated with higher circulating sCD14, a lower mean number of positive skin tests, and lower serum IgE levels in British and American white populations,5,9,26 and has also been found to be associated with non-atopic asthma and food allergy in various ethnic groups.15 Conversely, the CC genotype has been associated with higher levels of total serum IgE and a higher number of positive skin tests in a population from the Netherlands12,20,26 and the C allele has been associated with atopy, specifically to moulds, in a Czechoslovakian population.11
However, the associations between the C-159T polymorphism and atopic phenotypes have not always been consistent and, in contrast to these other findings, the CD14-159T allele was over-transmitted to atopic individuals in an inbred Hutterite population. Similarly, we have shown that the -159T allele was slightly more common in atopic adults. There was no association of this polymorphism with atopy in an Hispanic population,5 with atopic asthma in an Icelandic population,21 or with atopy and/or asthma in two German white populations.18,19
Although some populations have been relatively small, the allele frequencies of the C-159T polymorphism have not varied significantly between all populations investigated to date, with the exception of a Japanese population.9 The -159T allele frequencies ranged between 0.60 in a Japanese population,9 0.53 in a Chinese population,20 and 0.37 and 0.54 in all white populations: 0.37–0.41 in American;5,15 0.38 in Polish;16 0.42–0.46 in German;18,19 0.45 in Icelandic;21 0.47 in Australian; 0.52 in British;9 0.53 in Dutch;12 0.54 in Czechoslovakian.11 A meta-analysis of all populations studied to date revealed that there was no overall association between the C-159T polymorphism and asthma (p = 0.23) or atopy (p = 0.52); however, it did reveal significant between-study heterogeneity (p = 0.01).
It is difficult to explain the inconsistency of association studies for the C-159T polymorphism, even between populations of similar ethnicity. Environmental factors may differ between the study populations18 but, as has been highlighted by a number of authors, association studies are commonly difficult to replicate between different populations, especially when different phenotypic markers are analysed.18,19
There is tight linkage disequilibrium across the CD14 promoter region42 which would strongly favour linkage between C-159T and polymorphisms in CD14 or another gene on 5q, and may account for some of the associations seen to date. In support of this, the CD14-159T allele has been shown to be over-transmitted to atopic individuals in an inbred Hutterite population, only when on a haplotype with marker D5S642 previously shown to be linked to atopy in this population.43 It is therefore highly likely that the CD14-159T allele is actually in linkage disequilibrium with the susceptibility variant on 5q.10
It is also possible that the C-159T polymorphism has an age related influence on the development of atopy. The polymorphism has been associated with increased CD14 expression only in the serum of children,5,20 and there was no difference in serum CD14 levels or the expression of membrane bound levels of CD14 in a sample of adult blood donors with different CD14 genotypes.44 Additionally, a longitudinal study in Australian white subjects aged 8–25 years found that individuals with the CC genotype were more likely to have early onset atopy and early onset airway hyperresponsiveness, suggesting that the influence of -159C on the atopic genotype may be age specific.25 There is strong evidence to suggest that atopy and asthma are closely related entities, with a positive association between total serum IgE and number of positive skin tests, bronchial hyperresponsiveness, and development of asthma and doctor diagnosed asthma.45,46 Thus, alleles which are associated with atopy might be expected to be over-represented in individuals with asthma relative to the general population.12 However, to date, no association has been found between the C-159T polymorphism and atopic asthma. We have confirmed this in our population and in a meta-analysis of all populations studied, suggesting that separate genes on chromosome 5q may regulate susceptibility to high total serum IgE levels and bronchial responsiveness.5
Similarly, there are no publications showing an association between the CD14 C-159T polymorphism and asthma severity, although it has been suggested that it might modify the severity of airflow obstruction in asthmatics. A recent conference abstract reported a study conducted on 418 Australian adult asthmatics22 in which neither the CT nor TT genotypes were associated with life threatening asthma, although the TT genotype was found to be weakly associated with lower forced expiratory volume in 1 second (FEV1). However, there was no association between genotype and asthma severity (also defined by mean FEV1) in a smaller predominantly white American population.15 We have used a more extensive set of criteria to define asthma severity13 and have confirmed that there is no relationship between asthma severity and the C-159T polymorphism in our population.
ACKNOWLEDGEMENTS
The authors thank the patients for their participation in this study and the staff at Sir Charles Gairdner Hospital and the Asthma and Allergy Research Institute for all their assistance with patient recruitment.
FOOTNOTES
This project was funded by the Asthma and Allergy Research Institute, the Asthma Foundation of Western Australia, the NH&MRC and the Australian Medical Students Association. David Duffy is an NH&MRC Research Fellow.
REFERENCES
Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 1995;13:437–57.
Cleveland MG, Gorham JD, Murphy TL, et al. Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway. Infect Immun 1996;64:1906–12.
Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+T cells. J Immunol 1995;154:5071–9.
Romagnani S. Induction of TH1 and TH2 responses: a key role for the ‘natural’ immune response? Immunol Today 1992;13:379–81.
Baldini M, Lohman IC, Halonen M, et al. A polymorphism in the 5' flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am J Respir Cell Mol Biol 1999;20:976–83.
Peden DB, Tucker K, Murphy P, et al. Eosinophil influx to the nasal airway after local, low-level LPS challenge in humans. J Allergy Clin Immunol 1999;104:388–94.
Virchow JC Jr, Julius P, Matthys H, et al. CD14 expression and soluble CD14 after segmental allergen provocation in atopic asthma. Eur Respir J 1998;11:317–23.
Alexis N, Eldridge M, Reed W, et al. CD14-dependent airway neutrophil response to inhaled LPS: role of atopy. J Allergy Clin Immunol 2001;107:31–5.
Gao PS, Mao XQ, Baldini M, et al. Serum total IgE levels and CD14 on chromosome 5q31. Clin Genet 1999;56:164–5.
Ober C, Tsalenko A, Parry R, et al. A second-generation genomewide screen for asthma-susceptibility alleles in a founder population. Am J Hum Genet 2000;67:1154–62.
Buckova D, Holla LI, Schuller M, et al. Two CD14 promoter polymorphisms and atopic phenotypes in Czech patients with IgE-mediated allergy. Allergy 2003;58:1023–6.
Koppelman GH, Reijmerink NE, Stine CO, et al. Association of a promoter polymorphism of the CD14 gene and atopy. Am J Respir Crit Care Med 2001;163:965–9.
Kedda MA, Shi J, Duffy DL, et al. Characterisation of two polymorphisms in the LTC4 synthase gene in an Australian population of mild, moderate and severe asthmatics. J Allergy Clin Immunol 2004;113:889–95.
R Development Core Team. R: A language and environment for statistical computing. Vienna, Austria, 2003 ( http://www.R-project.org ).
Woo JG, Assa’ad A, Heizer AB, et al. The -159 C–>T polymorphism of CD14 is associated with nonatopic asthma and food allergy. J Allergy Clin Immunol 2003;112:438–44.
Lis G, Kostyk E, Sanak M, et al. . Pneumonol Alergol Pol 2001;69:265–72.
Gao PS, Mao XQ, Kawai M, et al. Negative association between asthma and variants of CC16(CC10) on chromosome 11q13 in British and Japanese populations. Hum Genet 1998;103:57–9.
Sengler C, Haider A, Sommerfeld C, et al. Evaluation of the CD14 C-159 T polymorphism in the German Multicenter Allergy Study cohort. Clin Exp Allergy 2003;33:166–9.
Heinzmann A, Dietrich H, Jerkic SP, et al. Promoter polymorphisms of the CD14 gene are not associated with bronchial asthma in Caucasian children. Eur J Immunogenet 2003;30:345–8.
Leung TF, Tang NL, Sung YM, et al. The C-159T polymorphism in the CD14 promoter is associated with serum total IgE concentration in atopic Chinese children. Pediatr Allergy Immunol 2003;14:255–60.
Hakonarson H, Bjornsdottir US, Ostermann E, et al. Allelic frequencies and patterns of single-nucleotide polymorphisms in candidate genes for asthma and atopy in Iceland. Am J Respir Crit Care Med 2001;164:2036–44.
Savirimuthu S, Yang IA, Shaw J, et al. CD14 polymorphisms and disease severity in asthmatics. Thoracic Society of Australasia and New Zealand. Adelaide, Australia. Respirology 2003;S2:A27.
Whitehead A, Whitehead J. A general parametric approach to the meta-analysis of randomized clinical trials. Stat Med 1991;10:1665–77.
LeVan TD, Bloom JW, Bailey TJ, et al. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity. J Immunol 2001;167:5838–44.
O’Donnell AR, Toelle BG, Marks GB, et al. Age-specific relationship between CD14 and atopy in a cohort assessed from age eight to twenty-five. Am J Respir Crit Care Med 2003;169:615–22.
Vercelli D, Baldini M, Stern D, et al. CD14: a bridge between innate immunity and adaptive IgE responses. J Endotoxin Res 2001;7:45–8.
Marsh DG, Neely JD, Breazeale DR, et al. Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 1994;264:1152–6.
Meyers DA, Postma DS, Panhuysen CI, et al. Evidence for a locus regulating total serum IgE levels mapping to chromosome 5. Genomics 1994;23:464–70.
Daniels SE, Bhattacharrya S, James A, et al. A genome-wide search for quantitative trait loci underlying asthma. Nature 1996;383:247–50.
Doull IJ, Lawrence S, Watson M, et al. Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness. Am J Respir Crit Care Med 1996;153:1280–4.
(CSGA ) Collaborative Study on the Genetics of Asthma. A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet 1997;15:389–92.
Noguchi E, Shibasaki M, Arinami T, et al. Evidence for linkage between asthma/atopy in childhood and chromosome 5q31–q33 in a Japanese population. Am J Respir Crit Care Med 1997;156:1390–3.
Hizawa N, Freidhoff LR, Ehrlich E, et al. Genetic influences of chromosomes 5q31–q33 and 11q13 on specific IgE responsiveness to common inhaled allergens among African American families. Collaborative Study on the Genetics of Asthma (CSGA). J Allergy Clin Immunol 1998;102:449–53.
Martinez FD, Solomon S, Holberg CJ, et al. Linkage of circulating eosinophils to markers on chromosome 5q. Am J Respir Crit Care Med 1998;158:1739–44.
Palmer LJ, Daniels SE, Rye PJ, et al. Linkage of chromosome 5q and 11q gene markers to asthma-associated quantitative traits in Australian children. Am J Respir Crit Care Med 1998;158:1825–30.
Lazarus R, Vercelli D, Palmer LJ, et al. Single nucleotide polymorphisms in innate immunity genes: abundant variation and potential role in complex human disease. Immunol Rev 2002;190:9–25.
Hagen G, Muller S, Beato M, et al. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 1994;13:3843–51.
Hata Y, Duh E, Zhang K, et al. Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 1998;273:19294–303.
Majello B, De Luca P, Lania L. Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem 1997;272:4021–6.
Birnbaum MJ, van Wijnen AJ, Odgren PR, et al. Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3. Biochemistry 1995;34:16503–8.
Dennig J, Hagen G, Beato M, et al. Members of the Sp transcription factor family control transcription from the uteroglobin promoter. J Biol Chem 1995;270:12737–44.
Vercelli D, Baldini M, Martinez F. The monocyte/IgE connection: may polymorphisms in the CD14 gene teach us about IgE regulation? Int Arch Allergy Immunol 2001;124:20–4.
Ober C, Tsalenko A, Willadsen S, et al. Genome-wide screen for atopy susceptibility alleles in the Hutterites. Clin Exp Allergy 1999;29 (Suppl 4) :11–5.
Heesen M, Blomeke B, Schluter B, et al. Lack of association between the -260 C–>T promoter polymorphism of the endotoxin receptor CD14 gene and the CD14 density of unstimulated human monocytes and soluble CD14 plasma levels. Intensive Care Med 2001;27:1770–5.
Sears MR, Herbison GP, Holdaway MD, et al. The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma. Clin Exp Allergy 1989;19:419–24.
Burrows B, Martinez FD, Halonen M, et al. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989;320:271–7.(M-A Kedda1,2,3, F Lose1,3)