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Genetic Variation in the Scavenger Receptor MARCO and Its Association with Chronic Obstructive Pulmonary Disease and Lung Infection in 10,604 IndividualsThomsen M.a, d · Nordestgaard B.G.a,b,d · Kobzik L.e · Dahl M.a,c,d
aDepartment of Clinical Biochemistry, Herlev Hospital, bThe Copenhagen City Heart Study, Bispebjerg Hospital, and cDepartment of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, and dFaculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; eDepartment of Environmental Health, Harvard School of Public Health, Harvard Medical School, Boston, Mass., USA Corresponding Author
Morten Dahl, MD, DMSc
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital
DK–2100 Copenhagen (Denmark)
Background: MARCO (macrophage receptor with collagenous structure) is a dominant receptor for unopsonized particles and bacteria in the lungs. Reduced function of this receptor due to genetic variation may be associated with susceptibility to chronic obstructive pulmonary disease (COPD) and lung infection. Objectives: To identify novel genetic variants in MARCO that are associated with reduced lung function, or increased risk of COPD or lung infection. Methods: We first screened 760 individuals with extreme lung phenotypes in a large general population study to identify novel variants in the MARCO gene. We next genotyped the entire cohort consisting of 10,604 individuals to assess the clinical relevance of these variants. Results: We identified 4 novel (R124H, K201N, P303L and G340W) and 5 previously described (H101Q, F282S, G319V, K387Q and E511D) non-synonymous variants. When screening the entire cohort for these variants, we found low minor allele frequencies ranging from 0.005 to 5%. None of the individual MARCO genotypes were associated with reduced lung function, or risk of COPD or lung infection. H101Q heterozygotes had an increased odds ratio for sepsis of 2.2 (95% CI: 1.1–4.4) compared to non-carriers, but none of the other MARCO genotypes were associated with the risk of sepsis. Conclusions: We identified 9 non-synonymous variants in the MARCO gene and showed that these variants are not major risk factors for COPD or lung infection in the Danish population. H101Q heterozygotes had increased sepsis risk, but further research is required to confirm this finding. This study is the first to examine genetic variants in MARCO and the risk of COPD and infections in humans.
© 2012 S. Karger AG, Basel
Scavenger receptors on the surface of alveolar macrophages are essential for the recognition and removal of inhaled particles and harmful airborne microorganisms . MARCO (macrophage receptor with collagenous structure ) has been shown to be a dominant receptor for unopsonized particles and bacteria in the lungs  and thus important for normal non-specific host defense [1,3]. This receptor is also upregulated in macrophages of the liver and spleen after infection [4,5,6] and is able to bind a high variety of ligands, including lipopolysaccharides and lipoteichoic acid as well as intact bacteria [1,7]. Reduced function of this receptor due to genetic variation may be associated with impaired pathogen clearance and increased susceptibility to chronic obstructive pulmonary disease (COPD) or lung infection.
In support of this hypothesis, mice with genetic deletion of MARCO demonstrate increased pulmonary inflammation in response to inhaled particulates and ozone compared to wild-type mice . Also, in a murine model of pneumococcal pneumonia, mice with genetic deletion of MARCO had impaired clearance of bacteria and diminished survival . After ovalbumin sensitization and aerosol challenge, MARCO-deficient mice also exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness, suggesting potential roles of MARCO in allergic asthma . Studies on genetic variability of MARCO in humans are scarce. Besides one recent study reporting an intronic SNP associated with an increased risk of pulmonary tuberculosis in a Chinese population , genetic variations in MARCO have not previously been examined in humans.
In this study, we tested the hypothesis that genetic variations in MARCO are associated with reduced lung function, COPD and lung infection. For this purpose, we first screened 760 individuals with extreme lung phenotypes in the Copenhagen City Heart Study to identify variants that potentially influence the risk of pulmonary disease in the general population. We next genotyped the entire cohort of the Copenhagen City Heart Study consisting of >10,000 individuals for the non-synonymous variants identified, and tested whether these genotypes were associated with reduced lung function, or increased risk of COPD or lung infection. As secondary aims, we also tested for associations with asthma and sepsis.
The Copenhagen City Heart study is a prospective general population study initiated in 1976–1978 with follow-up examinations in 1981–1982, 1991–1994 and 2001–2003 [12,13,14]. Individuals were selected on the basis of the national Danish Civil Registration System to reflect the population aged 20–100 years. The studies were approved by the Herlev Hospital and Danish ethical committees, and were conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants. At each examination, participants completed a questionnaire concerning their lifestyle factors, and physical parameters were measured. Blood samples were drawn for DNA extraction at the examinations performed in 1991–1994 and 2001–2003 on a total of 10,604 individuals.
To increase the likelihood of detecting genetic variations in MARCO associated with lung disease in the general population, we performed sequencing of the MARCO gene using the extreme phenotype approach . The risk populations with extreme phenotypes in this cohort were defined as those with the earliest onset of COPD and asthma, individuals with interstitial lung disease, individuals with the highest forced expiratory volume in 1 s [(FEV1) percent of predicted (FEV1%)] and the lowest FEV1% among smokers and non-smokers .
Fourteen PCR fragments were amplified, covering all 17 protein-coding exons of MARCO and the intron-exon boundaries. Mutational screening analysis of the amplicons was performed by LightScanner, a high-resolution DNA-melting curve analysis technique for variant detection . PCR fragments with DNA-melting curves differing from wild-type control DNA were subsequently sequenced. Primer sequences are listed in online supplementary table 1 (see www.karger.com?doi=10.1159/000342354 for all online suppl. material). We used a TaqMan-based assay to genotype the entire Copenhagen City Heart Study cohort for the mutations identified (Applied Biosystems Inc., Foster City, Calif., USA). Primers and probes for these analyses are listed in online supplementary table 2. Genotype results were confirmed by DNA sequencing of a subset sample.
FEV1 and forced vital capacity (FVC) were determined without inhalation of a bronchodilator using a dry wedge spirometer (Vitalograph; Maids Moreton, UK). Reference values for FEV1 and FVC were internally derived for men and women separately in a subsample of healthy never-smokers using multiple regressions with age and height as covariates [12,18]. If all individuals were used as reference rather than a subsample of never-smokers, the results were similar to those presented. Spirometry-defined COPD was FEV1/FVC <0.7 and FEV1 <80% of predicted . Information about hospitalization for COPD, pneumonia and sepsis was obtained by linking the participants to the national Danish Patient Registry and the national Danish Causes of Death Registry, using each participant’s unique Central Person Register number. Diagnoses were collected from the national Danish Patient Registry from 1976 through October 2011 and defined according to the 8th and 10th edition of the World Health Organization International Classification of Diseases. COPD was ICD8: 491–492; ICD10: J41–J44, pneumonia was ICD8: 480–486; ICD10: J12–J18, and sepsis was ICD8:38; ICD10: A40–A41, A49.9. Frequent pulmonary infection was defined as self-reported history of frequent pulmonary infections requiring medical attention during the last 10 years.
Statistical analyses were performed using STATA/SE version 11.1. Main effects of the genotype on FEV1%, FVC percent of predicted (FVC%) and FEV1/FVC were tested by Student’s t test. We used a logistic regression model adjusted for age, sex and pack-years of tobacco smoked to estimate odds ratios with 95% confidence intervals for the pulmonary and infectious endpoints. We tested for possible interactions between the MARCO genotype and different determinants of lung function in predicting FEV1%, FCV% and FEV1/FVC in ANCOVA models.
Clinical characteristics of the individuals with extreme lung phenotypes are listed in online supplementary table 3. As expected, individuals with early-onset COPD and interstitial lung disease were older and more likely to be current smokers than individuals without an extreme lung phenotype, whereas individuals with asthma were younger and less likely to be current smokers .
Sequencing of the MARCO gene in 760 individuals with extreme lung phenotypes identified a total of 42 genetic variations (table 1). Of these, 16 were in protein-coding regions and 9 were non-synonymous variations, of which R124H, K201N, P303L and G340W have not been described previously. Figure 1 shows a schematic view of the location of the 9 amino-acid-changing variants in relation to the previously described structure of MARCO . The F282S, G319V and G340W variants change polarity of the amino acid substituted and could be of functional relevance to MARCO. Also, E511D, which is located in the cysteine-rich domain required for ligand binding [3,20], could be of functional importance to the MARCO receptor.
We genotyped 10,604 individuals from the Copenhagen City Heart Study for all the 9 non-synonymous variants. Genotype frequencies and Hardy-Weinberg statistics are listed in table 2. Minor allele frequencies were low, ranging from 0.005 to 5%. All were in Hardy-Weinberg equilibrium. R124H, K201N, P303L, G340W and K387Q had minor allele frequencies <0.01% and were not further analyzed due to insufficient statistical power.
Individuals heterozygous for H101Q, F282S, G319V and E511D did not differ from non-carriers in FEV1%, FVC% or FEV1/FVC (fig. 2). Individuals homozygous for F282S had trends towards 4% increased FEV1%, 2% increased FVC% and 3% increased FEV1/FVC. Individuals homozygous for E511D had trends towards 3% reduced FEV1%, 6% reduced FVC% and 6% increased FEV1/FVC. However, none of these differences were statistically significant. We found no interaction between the MARCO genotypes and other determinants of lung function such as smoking status and gender. Also, combinations of the 4 variations in MARCO were not associated with differences in lung function.
In accordance with the results on lung function, multivariate adjusted odds ratios for spirometry-defined COPD and COPD hospitalization were not increased for any of the 4 MARCO variants analyzed (fig. 3). Individuals heterozygous for H101Q had odds ratios for spirometry-defined COPD and COPD hospitalization of 1.12 (95% confidence interval: 0.58–2.16) and 0.73 (0.35–1.58), respectively, compared with non-carriers. Corresponding odds ratios for F282S heterozygotes were 1.16 (0.93–1.44) and 1.09 (0.87–1.35), for G319V heterozygotes 1.00 (0.22–4.63) and 1.75 (0.48–6.40), and for E511D heterozygotes 1.07 (0.75–1.53) and 1.15 (0.82–1.60), respectively. F282S homozygotes had odds ratios for spirometry-defined COPD and COPD hospitalization of 1.31 (0.37–4.71) and 1.65 (0.53–5.17), respectively, compared with non-carriers. E511D homozygotes had an odds ratio for spirometry-defined COPD of 4.02 (0.29–55.4) compared with non-carriers. None of the 4 MARCO variants were associated with asthma (online suppl. fig. 1).
H101Q heterozygotes had an increased odds ratio for sepsis hospitalization of 2.16 (1.05–4.41; fig. 4). The odds ratios for self-reported frequent pulmonary infection and for pneumonia hospitalization in H101Q heterozygotes were 1.04 (0.66–1.65) and 0.63 (0.33–1.21), respectively. None of the other 3 MARCO variants were associated with self-reported frequent pulmonary infection, pneumonia hospitalization, sepsis hospitalization or a combination of these endpoints (fig. 4).
This is the first study to examine associations between genetic variations in MARCO and the risk of COPD and lung infections in humans. We identified 4 novel (R124H, K201N, P303L and G340W) and 5 previously described non-synonymous variants in the MARCO gene. None of these genetic variations in MARCO were associated with reduced lung function, or risk of COPD or lung infection. H101Q heterozygotes did have a 2-fold increased odds ratio for sepsis hospitalization compared to non-carriers, but further research is required to confirm this finding.
Current evidence indicating that reduced function of MARCO leads to impaired pathogen clearance and increased inflammation in the lungs comes from animal models [8,9]. These murine models use mice that are completely deficient in the expression of the gene, and this experimental setup does not necessarily mimic conditions found in humans where expression profiling has failed to detect increased MARCO expression in COPD patients . The genetic variants identified in this study may only be mildly deleterious or may not affect receptor function at all explaining our overall negative findings regarding lung disease. However, if the variants were functional as many rare non-synonymous variants are predicted to be , our negative results could be explained by decreased function of one scavenger receptor leading to compensatory increased expression of others . The structurally similar scavenger receptor A (SR-A) is another dominant receptor for unopsonized environmental particles and bacteria on human alveolar macrophages , and elevated expression of this receptor could perhaps partly compensate for MARCO deficiency. This receptor seems important in the development of COPD as we have previously found heterozygosity for a rare truncating mutation in the gene for SR-A to be associated with reduced lung function and increased COPD risk among men . Also, a recent study using a murine model exhibiting typical features of COPD found SR-A to play a key role in bacterial clearance and sustained inflammation, whereas MARCO seemed to be less important . Another explanation could be that in spite of our large sample size we do not have enough power to detect any potential association. However, using the same cohort, we have previously discovered a rare variant in the β2-adrenergic receptor with a minor allele frequency of 1% to be associated with reduced lung function and a 1.5-fold increased risk of COPD , so if a clinically relevant association existed for any of the MARCO variations identified it is unlikely that we would not have detected it. Beyond macrophage-driven anti-microbial/particle responses in the lung, MARCO could also have important roles in dendritic-cell-driven migration and function, implicating this receptor in allergic asthma in mice . Our results from humans did not indicate any association between the identified genetic MARCO variants and the risk of asthma.
We found that H101Q heterozygotes had an increased odds ratio for sepsis. However, this mutation did not associate with other infectious endpoints, and since the finding was based on a relatively low number of individuals it could represent a chance finding rather than a real phenomenon. On the other hand, studies support that MARCO [28,29] and other scavenger receptors [28,30,31,32] could be related to sepsis. The in vivo expression of MARCO is normally restricted to distinct populations of macrophages in the spleen and lymph nodes in mice. However, exposure of mice to BCG, lipopolysaccharides or bacterial sepsis rapidly upregulates MARCO expression in tissue macrophages, including dendritic cells and Kupffer cells of the liver [28,33,34]. A similar pattern of high MARCO expression was observed in the liver of 2 newborns who died of sepsis . Mechanistically, CD16 may promote Escherichia coli sepsis through an FcRγ-inhibitory pathway that prevents MARCO-mediated activities . Future research seems therefore warranted to elucidate the role of MARCO and other scavenger receptors in sepsis and to examine whether there are associations between the MARCO H101Q mutation and an increased risk of sepsis among humans.
Our present study is one of the first studies on genetic variations in MARCO in humans. All participants in this study were Danish whites, and although this eliminates any blurring due to ethnical heterogeneity of the study population, our results may not necessarily apply to other ethnicities. Bias caused by investigator knowledge of disease or risk factor status seems unlikely because our samples were selected from the general population and because genotyping of our samples was performed without investigator knowledge of disease status or lung function measurements. Recruitment from the general population could lead to a negative selection bias regarding the occurrence of functional variants in MARCO due to possible overrepresentation of healthy individuals. It is theoretically possible that individuals with genetic variations with profound consequences on receptor function were too sick to participate.
In spite of rapid advances in our knowledge of the human genome and increasing availability of investigative technology, the only gene consistently proven to influence COPD susceptibility is SERPINA1, encoding α1-antitrypsin [36,37,38]. Other potential risk genes for COPD include MMPs [39,40], SPB , ADRB2 [12,42], SERPINE2  and EPHX1 [42,43], but further research is required to verify the associations of these risk genes with COPD. Several genome-wide association studies have been conducted in pulmonary medicine, but the loci identified collectively account for only a small fraction of the observed hereditability in lung function and COPD, and have not been consistent across studies . Rare variants may explain some of the missing hereditability. This study identified 4 novel rare variants in the MARCO gene, and although we failed to find any association with disease, targeted resequencing of candidate genes in individuals with extreme phenotypes seems to be a valuable and cost-efficient approach to identify some of these rare variants .
In conclusion, we identified 4 novel and 5 previously described non-synonymous variants with low minor allele frequencies in the MARCO gene. By genotyping a large population-based study, we show that these variations are not major risk factors of reduced lung function, COPD or lung infection in the Danish population. Individuals heterozygous for H101Q did have an increased risk of sepsis hospitalization compared to non-carriers, but further research is required to confirm this finding. This study is the first to examine associations between genetic variants in MARCO and the risk of COPD and infections in humans.
This work was supported by the Herlev Hospital, Copenhagen University Hospital, the Danish Lung Foundation, the Danish Heart Foundation, the Copenhagen County Foundation and the University of Copenhagen (all from Denmark). The funding sources had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper and in the decision to submit it for publication.
The authors have no conflicts of interest to declare.
Morten Dahl, MD, DMSc
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital
DK–2100 Copenhagen (Denmark)
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