Ficolin‐2 (L‐ficolin), derived from the FCN2 gene, is an innate immunity pattern recognition molecule found in human serum in which inter‐individual variation in serum appears to be under genetic control. To validate and extend this finding, we developed a sandwich ELISA for detection of human Ficolin‐2 in serum samples and identified FCN2 genotypes with a Taq Man‐based minor groove binder assay and by sequencing. Serum samples were applied to gel‐permeation chromatography and fractions were analysed by an ELISA, SDS‐PAGE and subsequently Western blotting. In 214 Danish blood donors, the median Ficolin‐2 serum concentration was determined to 5.4 μg/ml (range: 1.0–12.2 μg/ml). An ELISA, SDS‐PAGE and Western blot analysis of gel‐permeation chromatography fractions showed that Ficolin‐2 comprises a mixture of covalently and non‐covalently linked Ficolin‐2 oligomers independent of the individual genotypes. The variation in serum concentration was associated with three polymorphisms in the promoter and one polymorphism in the structural part of the FCN2 gene. Further analysis indicated that two particular alleles on the same haplotype determined a low Ficolin‐2 concentration. Our results show that inter‐individual variation of Ficolin‐2 concentration is associated with polymorphisms in the promoter and the structural part of the FCN2 gene.

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.

No abstract