Non-synonymous polymorphisms in the FCN1 gene determine ligand-binding ability and serum levels of M-ficolin - PubMed (original) (raw)

Non-synonymous polymorphisms in the FCN1 gene determine ligand-binding ability and serum levels of M-ficolin

Christian Gytz Ammitzbøll et al. PLoS One. 2012.

Abstract

Background: The innate immune system encompasses various recognition molecules able to sense both exogenous and endogenous danger signals arising from pathogens or damaged host cells. One such pattern-recognition molecule is M-ficolin, which is capable of activating the complement system through the lectin pathway. The lectin pathway is multifaceted with activities spanning from complement activation to coagulation, autoimmunity, ischemia-reperfusion injury and embryogenesis. Our aim was to explore associations between SNPs in FCN1, encoding M-ficolin and corresponding protein concentrations, and the impact of non-synonymous SNPs on protein function.

Principal findings: We genotyped 26 polymorphisms in the FCN1 gene and found 8 of these to be associated with M-ficolin levels in a cohort of 346 blood donors. Four of those polymorphisms were located in the promoter region and exon 1 and were in high linkage disequilibrium (r(2)≥0.91). The most significant of those were the AA genotype of -144C>A (rs10117466), which was associated with an increase in M-ficolin concentration of 26% compared to the CC genotype. We created recombinant proteins corresponding to the five non-synonymous mutations encountered and found that the Ser268Pro (rs150625869) mutation lead to loss of M-ficolin production. This was backed up by clinical observations, indicating that an individual homozygote of Ser268Pro would be completely M-ficolin deficient. Furthermore, the Ala218Thr (rs148649884) and Asn289Ser (rs138055828) were both associated with low M-ficolin levels, and the mutations crippled the ligand-binding capability of the recombinant M-ficolin, as indicated by the low binding to Group B Streptococcus.

Significance: Overall, our study interlinks the genotype and phenotype relationship concerning polymorphisms in FCN1 and corresponding concentrations and biological functions of M-ficolin. The elucidations of these associations provide information for future genetic studies in the lectin pathway and complement system.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The structural and domain organization of M-ficolin and the organization of the exons in FCN1. A

M-ficolin oligomer consisting of 4 subunits each made of 3 identical polypeptides. B Structure of the M-ficolin polypeptide. White numbers indicate exon and dotted line indicate exons boundaries. The 5 non-synonymous SNPs encountered in the cohort are marked. Amino acid numbers include the signal peptide of 29 residues. C Representation of the promoter, exon and intron region of FCN1 drawn to scale. Exons are marked as boxes below the line and SNPs as lines above. All 26 SNPs genotyped in the cohort are marked.

Figure 2

Figure 2. Association between age and serum concentration of M-ficolin split by gender.

Full-drawn lines represents the estimated linear association for males (red) and females (black). Dotted lines represent 95% pointwise confidence intervals.

Figure 3

Figure 3. Correlation between the SNPs in the promoter region and exon 1 (R2 values).

R2 is given as percent. Stars indicate SNPs that are significantly associated with M-ficolin concentration.

Figure 4

Figure 4. Characterization of five recombinant M-ficolin proteins. A

The M-ficolin concentration measured in the supernatant of HEK293F cells transfected with plasmid encoding variants of M-ficolin. The wild type used as reference and the dotted line represents this value (100%). Boxes indicate range of data including median value. B Western blotting of supernatant from the wild type and the five variants of M-ficolin. For Ser268Pro also a lysate of the cells were used. The mutation for each variant is given beneath the lane. C Binding of recombinant M-ficolin to Streptococcus agalactiae serotype VI (GBS) The counts on the y-axis were obtained following incubation in GBS coated wells with recombinant M-ficolin and anti-M-ficolin antibody. Results displayed in A are from three while B and C are from two replicated experiments.

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