Human Molecular Genetics Advance Access originally published online on May 6, 2005
Human Molecular Genetics 2005 14(12):1651-1658; doi:10.1093/hmg/ddi173
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Polymorphisms in the FCN2 gene determine serum variation and function of Ficolin-2
1Department of Clinical Immunology, Tissue Typing Laboratory-7631, Rigshospitalet, 2100 Copenhagen, Denmark, 2Department of Biochemistry, Fukushima Medical University School, Fukushima 960-1295, Japan and 3Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
* To whom correspondence should be addressed at: Department of Clinical Immunology, Tissue Typing Laboratory-7631, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Tel: +45 35457631; Fax: +45 35398766; Email: garred{at}post5.tele.dk
Received March 16, 2005; Accepted April 26, 2005
DDBJ/EMBL/GenBank accession nos
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ABSTRACT |
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The ficolin 1, 2 and 3 (derived from the FCN1, 2 and 3 genes, respectively) are homologous soluble pattern recognition molecules of importance for innate immunity, comprising collagen-like and fibrinogen-like domains, binding to sugar groups on different types of microorganisms. Serum concentration of Ficolin-2 varies considerably in healthy individuals. Thus, we speculated whether this could be due to variations in the FCN2 gene. We sequenced the promoter region and the exons and intron–exon boundaries of FCN2 in Danish Caucasians. For comparison, FCN1 and FCN3 were also investigated. Ficolin-2 concentrations were measured in serum and the functional relevance of amino acid substituting polymorphisms in FCN2 was investigated by binding to and recovery from N-acetylglucosamine (GlcNAc). Both FCN1 and FCN2 contained polymorphisms in the promoters and structural parts of the genes, but only polymorphisms in FCN2 resulted in amino acid exchanges. FCN2 promoter polymorphisms were associated with marked changes in the Ficolin-2 serum concentration, whereas two polymorphisms clustered in the exon encoding the fibrinogen-like domain were associated with increased and decreased GlcNAc binding, respectively. In FCN3, only a single frame-shift deletion in exon 5 was detected. These results show that the FCN genes are polymorphic and that particularly FCN2 harbors functional polymorphic sites that regulate both the expression as well as the function of Ficolin-2, which may have pathophysiological implications for innate immunity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ficolins are soluble proteins of putative importance for host defense (1
). In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode Ficolin-1 (synonymous with M-ficolin and Ficolin/P35-related protein), Ficolin-2 (synonymous with L-ficolin, Ficolin/P35 and Hucolin) and Ficolin-3 (synonymous with H-ficolin, Hakata antigen and thermolabile ß2-macroglycoprotein), respectively. FCN1 and FCN2 are both located on chromosome 9q34, and are 80% homologous at the amino acid level, whereas FCN3 is assigned to chromosome 1 (1p36.11, 1p35.3) (2,3). On the amino acid level, Ficolin-3 reveals 
40% homology with both Ficolin-1 and Ficolin-2. FCN1 contains nine exons, whereas FCN2 and FCN3 are composed of eight exons. Ficolin-2 and Ficolin-3 are found in serum and exhibits inter-individual variation in serum concentrations (4–6). FCN2 is predominantly expressed in the liver and FCN3 in the liver and lung (2,7). Ficolin-1 is expressed in the lung, the spleen and by undifferentiated monocytes (2,8,9). Whether Ficolin-1 is present in serum is unknown. Thus far, Ficolin-1 has been found on the surface of mononuclear cells and it is uncertain whether it is secreted to the medium (10). The degree of expression of Ficolin-1 is down regulated upon monocyte maturation to macrophages.
The ficolins are synthesized as a single polypeptide containing N-terminal collagen-like and C-terminal fibrinogen-like sugar-binding domains, which are oligomerized into higher oligomeric forms comprising triple helix structures (1). The collagen-like multimeric structure is shared with C1q, mannose-binding lectin (MBL) and surfactants proteins A and D (SP-A and SP-D).
The fibrinogen-like domains of Ficolin-1 and Ficolin-2 have been shown to interact with carbohydrates, such as N-acetylglucosamine (GlcNAc) (10). Moreover, a general specificity for N-acetylated groups for Ficolin-2 has also been demonstrated (11). Both Ficolin-1 and Ficolin-2 appear to bind to different types of bacteria and Ficolin-2 may specifically bind to lipoteichoic acid from gram-positive bacteria (10,12). The ligands for Ficolin-3 are unknown, but distinct binding to certain strains of bacteria has been demonstrated (13). Ficolin-1 has been shown to enhance uptake of bacteria to monocytes and Ficolin-2 and Ficolin-3 have been shown to interact with the MBL-associated serine proteases enabling activation of the complement system (10,14,15). Taken together, these results provide evidence for the fact that the ficolins are important molecules in imparting innate immunity.
Because of the putative biological importance of these molecules in host defense, we studied the genetic variation in the FCN genes. We paid particular attention to the promoter and coding region polymorphisms of FCN2 to identify possible functional significance of these polymorphisms to FCN2 protein expression levels or functional ligand-binding activity.
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RESULTS |
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Identification of polymorphisms in the FCN genes
FCN1.
A total of 12 single nucleotide polymorphisms (SNPs) were identified in FCN1 by DNA sequencing analyses: five polymorphisms were detected in the promoter region, three silent polymorphisms in the coding region of exon 1, 6 and 8 and four in the exon–intron boundaries. The locations of the polymorphisms and genotype frequencies of the sequenced samples are shown in Figure 1 and Table 1. All polymorphisms adhered to the Hardy–Weinberg expectations (P>0.05).
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FCN2.
Five polymorphisms were identified in the promoter region of FCN2 and nine were found in the coding region by DNA sequence analyses, of which five were present in exons: two silent substitutions in exon 3 and 6 and three amino acid exchanging substitutions in exon 8, including a polymorphism that involves both a substitution and a deletion. These results show that Ficolin-2 polypeptide chains exist in at least three variants, which subsequently are named FCN2-A (wild-type), FCN2-B (Thr236Met) and FCN2-C (Ala258Ser). A fourth variant, FCN2-D (Ala264fs), was observed in one individual only. The frame-shift mutation Ala264fs results in sequence changes causing amino acid alterations in the C-terminal part of the Ficolin-2 polypeptide with a reduction of 39 amino acids before termination when compared with the wild-type. The locations of the polymorphisms and genotype frequencies of the sequenced samples are shown in Figures 1 and 2 and Table 1. All polymorphisms adhered to the Hardy–Weinberg expectations (P>0.05).
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FCN3.
Only one genotype variant was observed in the FCN3 gene: a frame-shift deletion at position +1637 in the exon 5 encoding the neck region, which was detected in one individual only. The location of the mutation and the genotype frequency are shown in Table 1 and Figure 2.
SNP identification.
SNP NCBI's GenBank accession numbers are given in Table 1. Nine novel SNPs were described in this study: three SNPs in FCN1, five SNPs in FCN2 and one FCN3 mutation. The SNPs which have not yet been given an ID number (rs...) in the NCBI SNP database (http://www.ncbi.nlm.nih.gov) are instead labeled with a submission number (ss...).
Haplotypes.
The promoter SNPs and SNPs causing amino acid changes were used to construct haplotypes. Using this strategy, we identified five common haplotypes in the FCN1 and FCN2, respectively, with frequencies >5% (Fig. 1). The observed haplotypes accounted for 87.0 and 79.5% of the chromosomes, respectively. The results presented in Table 2 shows that several of the SNPs are in significant pairwise linkage disequilibrium (LD).
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FCN2 promoter polymorphisms influence the Ficolin-2 serum concentration
The level of Ficolin-2 in serum from 76 normal individuals varied considerably from 0.72 to 6.0 µg/ml with a median concentration of 3.0 µg/ml. The variation in Ficolin-2 serum concentration was markedly and statistically significantly associated with the presence of polymorphisms in the promoter region of FCN2 at positions –986 (P=0.0005), –602 (P=0.02) and –4 (P=0.01), respectively, whereas no association was seen with the polymorphisms at positions –557 (P=0.45) and –64 (P=0.25), respectively (Fig. 3), or the polymorphisms located in the coding region (data not shown). The genotypes that were associated with variation in Ficolin-2 serum concentration revealed a phenotype that was gene dose-dependent, i.e. homozygotes had either the highest or the lowest Ficolin-2 concentrations, whereas heterozygotes had intermediate concentrations. The inferred haplotypes did not provide additional information.
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Binding of Ficolin-2 variants towards GlcNAc
To investigate putative differences in the binding of the Ficolin-2 variants to GlcNAc, dilutions of whole human serum comprising the different homozygous variants of Ficolin-2 were added to ELISA wells coated with GlcNAc–bovine serum albumin (BSA) and captured Ficolin-2 was detected with an anti-Ficolin-2 antibody. The amount of bound Ficolin-2 was compared with the total serum level of Ficolin-2. The actual Ficolin-2 serum concentration within each genotype did not influence the Ficolin-2 binding profile to GlcNAc–BSA. Differences in binding capacity were observed for the three investigated Ficolin-2 variants, indicating functional relevance of the polymorphisms on Ficolin-2 binding affinity towards GlcNAc (Fig. 4A). The FCN2-C (Ala258Ser) variant showed larger binding capacity when compared with the FCN2-A genotype (wild-type), whereas variant FCN2-B (Thr236Met) revealed a reduced binding capacity when compared with FCN2-A. In control experiments, no binding was observed for any of the serum variants to BSA-coated wells.
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Affinity purification with GlcNAc-beads and recovery
Whole human homozygous serum Ficolin-2 variants were incubated with GlcNAc-beads and Ficolin-2 was eluted with GlcNAc. The purified- and total-serum Ficolin-2 concentrations were measured by sandwich ELISA using the two monoclonal anti-Ficolin-2 antibodies, GN4 and the biotinylated GN5 as described in the Materials and Methods section. Differences in binding capacity were observed for the three Ficolin-2 variants investigated. The FCN2-C allele showed a marked increase in binding capacity to GlcNAc when compared with the FCN2-A variant (Fig. 4B), whereas FCN2-B showed reduced purification yield. The actual Ficolin-2 serum concentration within each genotype did not influence the purification yield of Ficolin-2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous serum-based studies have shown that the concentration of Ficolin-2 varies considerably among individuals (4
,16). Thus, we speculated whether this variation could be genetically determined by variation in the promoter region of the FCN2 gene or in the structural gene itself. In agreement with a previous Scottish study, we found the median Ficolin-2 serum concentration in Caucasians to be 3.0 µg/ml (4). Five polymorphic sites were identified by DNA sequencing of the promoter region in an initial screening analysis, which subsequently was extended with additional 96 individuals. The polymorphisms at positions –986, –602 and –4 all significantly correlated with either decrease or increase in the Ficolin-2 concentration in a gene dose-dependent manner, whereas no association with variation in serum concentration was seen for the polymorphisms at positions –557 or –64 or the amino acid substitutions in exon 8 (data not shown). The protein concentration varied about 2-fold due to the different polymorphisms. For the alleles at positions –602 and –4, it was evident that it was the minority alleles (allele frequencies 0.202 and 0.24, respectively) that were associated with the highest serum concentration, whereas at position –986, the allele frequency of the allele associated with the highest serum concentration was 0.51. Further research using reporter gene assays and electrophoretic mobility shift assays will hopefully provide more detailed information about the molecular mechanisms behind the observed differences in Ficolin-2 expression. The observed differences in FCN2 were found in healthy individuals. It remains to be seen whether differential FCN2 expression would be observed in individuals during acute phase reactions or different disease processes, either of which might lead to altered transcription. Of particular interest were the two polymorphisms we observed in exon 8 in the fibrinogen-like domain of FCN2, which resulted in amino acid substitutions. The allelic variant at amino acid 236 leading to an exchange of a threonine with a methionine (named variant FCN2-B, allele frequency of 0.15) markedly decreased the binding capacity of Ficolin-2 to GlcNAc conjugated to agarose beads in homozygous variants compared with the wild-type. Conversely, the allelic variant at amino acid 258 leading to an exchange of alanine with serine (named variant FCN2-C, allele frequency of 0.061) markedly increased the binding capacity of Ficolin-2 using the assay system described earlier. In addition, a third variant was observed in one person only (FCN2-D), which was a frame-shift mutation in amino acid position 264 (Ala264fs) resulting in alterations in the C-terminal part of the Ficolin-2 polypeptide with a reduction of 39 amino acids before termination compared with the wild-type. If this is a true low frequency polymorphism, it may be assumed that it may give rise to a Ficolin-2 deficiency state in homozygotes due to incorrect folding of the molecule. Several silent polymorphisms were observed in exons 3 and 6 as well as base substitutions in exon–intron boundaries, but these were not subjected to further analyses.
Including the promoter and structural alleles showed that the FCN2 gene locus comprised five major haplotypes with frequencies ranging from 7.8 to 34.2%. These haplotypes accounted for 79.5% of all the chromosomes. Pairwise investigation between SNPs indicates LD with varying degree. The importance of FCN2 haplotypes for Ficolin-2 serum concentration and function is at present unknown. However, variation in the serum concentration as well as our functional analyses were associated more with the distinct SNPs observed than were the inferred haplotypes, suggesting that they may be loci of biological relevance.
The FCN2 genetic system is apparently regulated on the transcriptional level both through promoter polymorphisms as well as through structural polymorphisms. Whether these alleles may be associated with clinical pathophysiology and/or disease susceptibility remains to be established. The first hint of such a possibility has recently been reported because low concentrations of Ficolin-2 have been found with increased frequency in children with recurrent respiratory infections when compared with controls (17). However, it is striking that all the three amino acid changes in FCN2 are clustered in close vicinity in exon 8 encoding the fibrinogen-like domain (illustrated in Fig. 2), which could indicate that the relatively high frequency of these amino acid alterations as well as the promoter alleles influencing the Ficolin-2 serum concentration have arisen due to positive selection. Thus, the genetically determined variation in the Ficolin-2 concentration and change in binding affinity or specificity may have conferred some selective advantages in host–pathogen interactions analogous to what has been previously suggested for the MBL2 genetic system (18–20).
When we sequenced the exons and exon–intron boundaries as well as the promoter region of the FCN1 gene, we found that both the promoter as well as the structural regions harbored several polymorphic sites, but in contrast to FCN2, no alternations causing amino acid changes were found. Because FCN1 is abundantly expressed in peripheral monocytes (21), further characterization of the putative influence that these promoter alleles may have on the FCN1 expression level can ideally be investigated further by, e.g. real-time PCR-based techniques. The promoter SNPs in the FCN1 gene locus comprises five major haplotypes with frequencies ranging from 7.6 to 30.9%, accounting for 87% of the chromosomes.
No polymorphisms were observed when we sequenced the FCN3 gene. Consistent with this finding are the investigations of several thousands of individuals from different ethnic groups showing that deficiency of Ficolin-3 antigen (Hakata antigen) is an extremely rare condition (22). However, the Ficolin-3 concentration is highly variable in systemic lupus erythematosus patients due to autoantibodies against the protein. Nevertheless, we found a deletion mutation in one of the investigated individuals creating a premature stop codon in exon 5. Whether this is a de novo mutation or an established mutation in the Caucasian population remains to be seen, but in the homozygous situation, the resulting molecule, if it is assembled, will be truncated and not be able to participate in ligand binding. We did not choose to sequence the promoter region of FCN3 at this stage; however, variation in Ficolin-3 serum concentration has been observed in normal individuals (7–23 µg/ml), which could be genetically determined (6).
In conclusion, no polymorphisms causing amino acid changes were found in the FCN1 and FCN3 genes, except for one mutation observed in one individual introducing a stop codon in the FCN3 gene. Further studies are required to determine the relevance of the promoter polymorphisms detected in the FCN1. The promoter region of FCN2 harbor polymorphic sites that are associated with variation in the Ficolin-2 serum concentration and polymorphisms in the exon 8 encoding the fibrinogen-like domain cause amino acid changes that affect the binding of Ficolin-2 to GlcNAc. These alleles may predispose individuals to different infectious and autoimmune conditions.
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MATERIALS AND METHODS |
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Subjects
Peripheral venous blood samples were obtained from 157 unrelated adult Danish Caucasians. The Local Ethics Committees approved the study. Genomic DNA was prepared from each blood sample using the method described by Miller et al. (23
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DNA sequencing of the FCN genes
Direct sequencing of the promoter regions of the FCN1 and FCN2 genes, spanning from position –1325 to –1 bp and –1375 to –1 bp, respectively, as well as all exons and intron–exon boundary sequences was performed on genomic DNA templates from 60 and 157 individuals, respectively. In addition, all the exons and intron–exon boundary sequences of the FCN3 gene were sequenced from 60 individuals. The promoter regions were amplified by PCR in four and five overlapping PCR fragments, respectively, and the complete coding regions of each exon of the FCN genes were amplified using primers designed from flanking intronic or untranslated sequences. Each fragment was amplified by using a single primer set (Table 3), where the forward primers contained a 5'-T7 sequence (5'-ttatacgactcacta-3'). PCR amplifications were carried out in 20 µl volumes containing: 50 ng genomic DNA, 0.25 µM of each primer, 2.5 mM MgCl2, 0.2 mM dNTP, 50 mM KCl, 10 mM Tris–HCl, pH 8.4 and 0.4 units of Platinum Taq DNA polymerase (Invitrogen). The PCR reactions were performed at the following cycling parameters: 2 min 94°C, 35 cycles (30 s 94°C, 60 s 58°C, 60 s 72°C), 5 min 72°C and were sequenced in the forward direction using the ABI BigDye cycle sequencing terminator kit (Applied Biosystems, Foster City, CA, USA). Sequence reactions were purified using streptavidin beads using 5'-biotinylated T7 sequence primers (GenoVision). Sequence analysis was performed on an ABI Prism 3100 Genetic Analyser (Applied Biosystems). The resulting DNA sequences were aligned using BioEdit software, and DNA polymorphisms were confirmed visually from sequence electropherograms. Additionally, the FCN2 promoter fragments, nos 1, 2 and 4, and the FCN2 exon 8 fragment were sequenced in 96 and 97 individuals, respectively, as described earlier. DNA sequences containing promoter and structural polymorphisms/mutations of the FCN1, FCN2 and FCN3 genes have been submitted to NCBI's GenBank.
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Determination of the Ficolin-2 serum concentration by ELISA
Ficolin-2 concentrations were measured in 76 individuals as previously described (16
). Briefly, microtiter plates (Maxisorb, Nunc) were coated with 100 µl monoclonal anti-Ficolin-2 antibody (GN4, 2.5 µg/ml) in phosphate-buffered saline (PBS) and incubated overnight at 4°C and the wells were washed in PBS containing 0.05% Tween-20 (PBS–T). Serum samples were diluted (1:50) in PBS–T in a volume of 100 µl. A standard dilution series (1:10–1:1280) of a human serum pool in PBS–T were added to the plates. A positive control of recombinant Ficolin-2 (4 µg) and a negative control without Ficolin-2 were included and the plates were incubated overnight at 4°C. The wells were washed and 100 µl of biotinylated monoclonal anti-Ficolin-2 antibody (GN5, 2.5 µg/ml) in PBS–T was added and incubated for 1 h at 37°C. Peroxidase-conjugated streptavidin diluted (1:5000) in PBS-T was added, and incubated for 1 h at 37°C. After washing, the plates were developed with OPD tablets (DAKO) and the absorbance was measured at 490 nm.
Binding of Ficolin-2 to GlcNAc
Microtiter plates (Maxisorb, Nunc) were coated with 100 µl GlcNAc–BSA (Mobitec, UK; 4 µg/ml in PBS per well) or BSA and incubated overnight at 4°C. Plates were washed three times in PBS–T and blocked for 2 h in PBS containing 2% BSA (Sigma). Dilutions of human serum variants (1:1–1:750) in PBS–T, from each variant from three patients not included in the current characterization of the FCN polymorphisms were added to the wells and incubated overnight at 4°C. Subsequently, the plates were incubated for 1 h at 37°C with 100 µl of the monoclonal anti-Ficolin-2 antibody, GN5 (23 µg/ml) followed by incubation with HRP-conjugated anti-mouse antibody (0.65 µg/ml) at 37°C for 1 h. The reaction was developed with OPD tablets (DAKO) and the absorbance was measured at 490 nm. After each incubation step, the wells were washed three times in PBS–T.
Affinity purification with GlcNAc-beads
Whole sera containing Ficolin-2 structural variants (three individuals of each variant) were purified with GlcNAc–agarose beads (Sigma, USA). A total of 500 µl human serum was incubated with 50 µl GlcNAc-beads and 500 µl binding buffer (50 mM Tris–HCl, 200 mM NaCl, 20 mM EDTA, pH 7.8) overnight at 4°C under rotation. The samples were applied to a microcentrifuge column and washed three times in 500 µl binding buffer, and protein was eluted with 400 mM GlcNAc. Ficolin-2 concentrations in full serum and eluates of purified Ficolin-2 were measured for each genotype variant by the ELISA assay described earlier.
Statistics
The inferred haplotypes and LD, expressed as D' quantified between all pairs of biallelic loci, were estimated using the SNPAlyze program version 4.0 (Dynacom, Yokohama, Japan). The significance of association was determined by contingency table analysis using chi-square or Fisher's exact test when the expected number of individuals was below 5. Hardy–Weinberg equilibrium was analyzed using gene frequencies obtained by simple gene counting and the chi-square test with Yates' correction for comparing observed and expected values. Non-parametric Kruskall–Wallis or Mann–Whitney tests for unpaired group comparisons were used to evaluate the effect of the FCN2 promoter polymorphisms on Ficolin-2 serum concentration. All analyses were two-tailed.
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ACKNOWLEDGEMENTS |
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Excellent technical assistance was provided by Ms Vibeke Witved and Vibeke Weirup. Grant support was obtained from the Danish Medical Research Council, The Novo Nordisk Foundation, Copenhagen Hospital Corporation Research Foundation and Rigshospitalet. T.H. is a Copenhagen University research fellow.
Conflict of Interest Statement. None declared.
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FOOTNOTES |
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The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 
ss32469536, ss32469537, ss32469543, ss32469544, ss32469546, ss32469547, ss37043610, ss37043612 and ss37043613.
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