Abstract
L-ficolin (ficolin-2) is a complement-activating pattern-recognition lectin taking part in the innate immune response. Both its serum concentration and sugar binding capacity are influenced by single nucleotide polymorphisms (SNP) of the corresponding FCN2 gene. Cost-effective and simple procedures, based on polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism for an investigation of four FCN2 SNPs are proposed: −64 A > C (rs7865453), −4 A > G (rs17514136; both located in the promoter region), +6359 C > T (rs17549193), +6424 G > T (rs7851696; both in exon 8). Variant alleles of −64 and +6424 (in strong linkage disequlibrium) are known to be associated with low L-ficolin level or activity. In contrast, variant alleles at positions −4 and +6359 (also in strong linkage disequlibrium) correspond to higher values. Since several L-ficolin clinical associations have been reported, FCN2 genotyping seems to be a valuable tool for disease association studies.
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Introduction
Ficolins are complement-activating pattern-recognition molecules taking part in the innate immune response. They are oligomeric lectins composed of basic subunits (each a trimer of identical polypeptide chains). The cysteine-rich N-terminus enables forming of disulphide bonds, determining the quaternary structure. It is followed by a collagen-like region, responsible for binding of mannan-binding lectin-associated serine proteases (MASPs). At the C-terminus, a fibrinogen-like domain is present, which binds target structures. Three ficolins have been identified in humans: M-ficolin (ficolin-1), L-ficolin (P35, ficolin-2), and H-ficolin (Hakata antigen, ficolin-3), encoded by FCN1, FCN2, and FCN3 genes, respectively (Matsushita 2010). L-ficolin is expressed mainly in hepatocytes (Matsushita et al. 1996) and secreted into the bloodstream (Kilpatrick et al. 1999; Hummelshoj et al. 2005). It acts as an opsonin and initiates, in cooperation with MASPs, activation of complement via the lectin pathway. Like other proteins of that family, it recognizes N-acetyl-d-glucosamine and related structures (Matsushita et al. 1996; Kilpatrick et al. 1999; Hummelshoj et al. 2005).
The FCN2 gene (GenBank accession no. NG_011649) is located on chromosome 9 (9q34). It contains eight exons and seven introns. The first exon encodes 5′UTR, signal peptide (25 amino acid residues) and nine N-terminal amino acids of the mature protein; the second and third exons are responsible for synthesis of the collagen-like domain; the fourth encodes the linker peptide between collagen-like and fibrinogen-like domains; and exons 5–8 encode the fibrinogen-like domain. Exon 8 also encodes 3′UTR (Endo et al. 1996). This gene exhibits considerable genetic polymorphism affecting the promoter region, exons and introns (Hummelshoj et al. 2005; Herpers et al. 2006; Cedzynski et al. 2007). Five promoter single nucleotide polymorphisms (SNPs), at positions: −986 (A > G, rs3124952), −602 (G > A, rs3124953), −557 (A > G, rs3811140), −64 (A > C, rs7865453), −4 (A > G, rs17514136) from ATG as well as three located in exons (+2488 T > C, exon 3, rs4520243; +6359 C > T, exon 8, rs17549193; +6424 G > T, exon 8, rs7851696) have been reported to be quite common among Caucasians (Hummelshoj et al. 2005; Herpers et al. 2006). The last two mentioned result in amino acid substitutions: Thr236Met and Ala258Ser, respectively. A rare (found in one case only) 6442–6443delCT > A frame-shift mutation (Ala264fs) leads to the shortening of polypeptide chain by 39 amino acid residues (Hummelshoj et al. 2005; Table 1). Additionally, Hummelshoj et al. (2008) described 14 promoter and 22 coding region FCN2 polymorphisms in various ethnical groups.
Several studies involving Caucasians (Danish, Polish) revealed that 3 promoter variations (−986, −602, −4) influence serum L-ficolin concentrations while two variants in exon 8 (+6359 C > T, exon 8; +6424 G > T)—the sugar-binding capacity of the protein (Hummelshoj et al. 2005; Munthe-Fog et al. 2007; Cedzynski et al. 2007). Among those mentioned, there are two pairs of SNPs in strong linkage disequilibrium: −64/+6424 and −4/+6359. Relatively low L-ficolin levels were associated with variant alleles for −64/+6424 while high, for −4/+6359, respectively (Munthe-Fog et al. 2007; Cedzynski et al. 2007). Since several L-ficolin clinical associations have been reported (Kilpatrick and Chalmers 2012), FCN2 genotyping seems to be a valuable tool for disease association studies.
Methods for FCN2 genotyping previously published were based on sequencing, reverse hybridization technology or real-time PCR with tagged probes (Ojurongbe et al. 2012; Munthe-Fog et al. 2007; Cedzynski et al. 2007; Haerynck et al. 2012). We propose new, simple and cost-effective genotyping of SNPs in the FCN2 gene, at sites −64, −4, +6359, and +6424. Our methods employ simple allele-specific PCR or restriction fragment length polymorphism (RFLP)-PCR procedures.
Material and methods
Blood samples and DNA extraction
Samples of peripheral blood were obtained from healthy unrelated Polish adults (n = 102) with their informed consent. The study was approved by the local ethical committee. The genomic DNA was extracted using GeneMATRIX Quick Blood DNA Purification Kit (EURx, Poland), according to manufacturer’s protocol.
FCN2 genotyping
Designing of primers
PRIMER3 software (available online at site http://workbench.sdsc.edu) has been employed to design all primers used in this work.
Investigation of −64 A > C (rs7865453) polymorphism
For an investigation of −64 A > C polymorphism, two separate amplifications for each sample, with 0.4 μM of reverse primers, specific for A and C variants (P64_RA, PCR I; P64_RC, PCR II) and 0.2 μM common forward primer (P64_F), were performed (Table 2, Fig. 1). To enhance the specificity of the P64_RA primer, the substitutions of two nucleotides three and four bases before the 3′ end were introduced (Table 2, underlined). To each reaction mix, an additional reverse primer (P64_Ctrl; 0.1 μM) was added as an internal control of PCR. Each DNA sample (100 ng) was added to a reaction volume of 25 μl containing primers, 2.5 μl of 10× polymerase buffer, 1 U of Taq DNA polymerase (Fermentas, Lithuania), 2 mM MgCl2 and 200 μM deoxyribonucleoside triphosphate mix (dNTPs, Invitrogen, USA). Reactions were run using a C1000 Thermal Cycler (Bio-Rad, USA) under the following conditions: 95 °C for 3 min, then 35 cycles of 95 °C for 30 s, 62 °C for 30 s, 72 °C for 15 s, and finally 72 °C for 5 min (final elongation). The PCR products were analyzed on a 6 % polyacrylamide gel.
Investigation of −4 A > G (rs17514136) polymorphism
For an investigation of −4 A > G polymorphism, a simple PCR-RFLP procedure was employed. Each DNA sample (100 ng) was added to a reaction volume of 25 μl, containing 2.5 μl of 10× polymerase buffer, 1 U of Taq DNA polymerase (Fermentas), 2 mM of MgCl2, 200 μM of dNTP mix (Invitrogen) and 0.4 μM of primers P4_F and P4_R (Table 3). Reactions were run using a C1000 Thermal Cycler (Bio-Rad) under the following conditions: 95 °C for 3 min, then 35 cycles of 95 °C for 30 s, 63 °C for 30 s and 72 °C for 15 s, followed by a final elongation step (72 °C for 5 min). After that, the PCR products (5 μl) were treated with 1 U of MboII enzyme (Fermentas), at 37 °C for 2 h. The digestion products were further analyzed on a 6 % polyacrylamide gel (Fig. 2).
Investigation of +6359 C > T (rs17549193) polymorphism
The rs17549193 (+6359 C > T) polymorphism was investigated with the help of two separate PCRs, with specific primers for C or T variants (Table 4, Fig. 3). PCR I, enabling C allele recognition was performed with primers P6359_F1 and P6359_R1 (0.4 μM each). P6359_R2 primer (0.08 μM) was added as an internal PCR control. Consequently, PCR II (for T variant) was performed with the same amounts of P6359_F2, and P6359_R2 primers, and P6359_F1 as an internal control (Fig. 3).
P6359_R1 and P6359_F2 primers were designed to be specific for C and T variants, respectively. To enhance their specificity, we introduced a substitution of single nucleotide six bases before 3′ end of the first mentioned and four bases before 3′ end of the second (Table 4, underlined). Each DNA sample (100 ng) was added to a reaction volume of 25 μl containing 2.5 μl 10× polymerase buffer, 1 U of Taq DNA polymerase (Fermentas), 2 mM MgCl2, 200 μM dNTP mix (Invitrogen) and primers, as described above. PCRs were performed on a C1000 Thermal Cycler (Bio-Rad) under the following conditions: 95 °C for 3 min, then 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 15 s, and finally 72 °C for 5 min (final elongation). Products were analyzed on a 6 % polyacrylamide gel.
Investigation of +6424 G > T (rs7851696) polymorphism
For an investigation of +6424 G > T polymorphism, two separate amplifications for each sample, with 0.4 μM of forward primers, specific for G and T variants (P6424_FG, PCR I; P6424_FT, PCR II) and common reverse primer (P6424_Rev), were performed (Table 5, Fig. 4). To enhance the specificity of FG and FT primers, substitutions of single nucleotides three bases before the 3′ end were introduced (Table 5, underlined). To each reaction mix, an additional forward primer (P6424_Ctrl; 0.1 μM) was added as an internal control of PCR. Each DNA sample (100 ng) was added to a reaction volume of 25 μl containing primers, 2.5 μl of 10× polymerase buffer, 1 U of Taq DNA polymerase (Fermentas), 2 mM MgCl2 and 200 μM dNTP mix (Invitrogen). Reactions were run using C1000 Thermal Cycler (Bio-Rad) in the following conditions: 95 °C for 3 min, then 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 15 s, and then finally 72 °C for 5 min (final elongation). PCR products were analyzed on a 6 % polyacrylamide gel.
Direct sequencing
All of the samples genotyped by RFLP-PCR or PCR methods were reanalyzed by direct sequencing of the respective DNA fragments. First, PCRs were run on a C1000 Thermal Cycler (Bio-Rad), using appropriate spanning primers (Table 6), under the following conditions: 95 °C for 3 min, then 35 cycles (95 °C for 30 s, 60 °C for 30 s, 72 °C for 15 s), and finally 72 °C for 5 min (final elongation). An exception was −4 A > G (rs17514136) polymorphism where 63 °C instead of 60 °C was used.
The PCR products were purified with the help of Wizard SV Gel and PCR Clean-Up System (Promega, USA). Samples thus prepared (1 μl) were directly used as templates for sequencing, performed using the GeneAnalizer-3000 sequencer (Applied Biosystems, USA) and BigDye Terminator kit v. 3.1 (Applied Biosystems), according to the manufacturer’s instructions.
Results
Investigation of −64 A > C (rs7865453) polymorphism (FCN2 promoter region)
The −64 A > C SNP was investigated by two separate PCR reactions (both giving products of length of 207 bp), with specific primers recognizing the A or C variant. An additional primer (P64_Ctrl) was added to each reaction mix, as an internal control, yielding a 399 bp product, visible after electrophoresis independently of genotype. In consequence, for wild-type homozygotes (A/A), electrophoresis revealed two bands (specific, 207 bp; control, 399 bp) after PCR I and one after PCR II (control). Two bands after analysis of products of both PCRs correspond to the A/C heterozygous variant while one after PCR I and two after PCR II correspond to the C/C homozygote (Fig. 5a). The results of direct sequencing of the same samples are presented in Fig. 5b.
Investigation of −4 A > G (rs17514136) polymorphism (FCN2 promoter region)
This SNP was investigated by a simple PCR-RFLP method, employing MboII endonuclease. The A variant has two digestion sites while substitution with G at site −4 leads to the disappearance of one of them (Fig. 2). Consequently, the PCR product (296 bp) corresponding to the majority allele was cleaved into three fragments (128, 88, and 80 bp). The variant G allele yields two (208 and 88 bp) digestion products (Table 3). Therefore, electrophoresis of samples coming from A/A homozygotes gives three bands, A/G heterozygotes gives four (208, 128, 88, 80 bp), and G/G homozygotes two bands of length 208 and 88 bp (Fig. 6a). The results of direct sequencing of the same samples are presented in Fig. 6b.
Investigation of +6359 C > T (rs17549193) polymorphism (FCN2 exon 8)
Two separate PCRs for each sample were performed to investigate this SNP. Independently of genotype, a band corresponding to internal control (259 bp) is observed after electrophoresis of the PCR products. Allele-specific bands for C (173 bp) and T (127 bp) variants are visible after PCR I or II only, respectively (Fig. 7a). Finally the C/C genotype results in two bands (specific and control) after separation of products of the first PCR and one (control) band after the second. Two bands for both PCRs correspond to C/T heterozygotes, while one for PCR I and two for PCR II correspond to T/T variant homozygotes (Fig. 7a). The same samples were also sequenced to confirm PCR results (Fig. 7b).
Investigation of +6424 G > T (rs7851696) polymorphism (FCN2 exon 8)
Two separate PCR reactions (both giving products of length of 275 bp) were performed, with specific primers recognizing either the G or T variant. An additional primer (P6424_Ctrl) was added to each reaction mix as an internal control, yielding in 358 bp product, visible after electrophoresis independently of genotype. Consequently, wild-type homozygotes (G/G), revealed two bands (specific, 275 bp; control, 358 bp) after PCR I and one after PCR II (control). Two bands after analysis of products of both PCRs correspond to G/T heterozygous variant while one after PCR I and two after PCR II correspond to the T/T homozygote (Fig. 8a). The results of direct sequencing of the same samples are presented in Fig. 8b.
Discussion
The FCN2 gene polymorphisms studied, which influence both L-ficolin levels and its sugar-binding capacity, are considered to be clinically significant. For example, it was shown that the +6359 C > T variant allele is a risk factor for staphylococcal peritonitis in continuous ambulatory peritoneal dialysis patients (Meijvis et al. 2011) as well as bacterial infections following orthotopic liver transplantation (de Rooij et al. 2010). Heterozygosity for SNPs −64 A > C and +6424 G > T was associated with earlier onset of Pseudomonas aeruginosa colonization in cystic fibrosis (Haerynck et al. 2012). Moreover, an interplay between liver donor’s and recipient’s FCN2 +6424 genotype may influence the risk of cytomegalovirus infection in the latter (de Rooij et al. 2011). Homozygosity for the variant (T) allele seemed to protect from cutaneous leishmaniasis (Assaf et al. 2012). The minority alleles at positions −4 (G) and −986 (A, not studied here) were associated with a higher susceptibility to schistosomiasis (Ouf et al. 2012). Hoang et al. (2011) found the AGGG haplotype (corresponding to SNPs at positions: −986, −602 (both not investigated in this report), −4 and +6424) to be protective against hepatitis B and hepatocellular carcinoma.
Methods used so far for FCN2 genotyping have been based mainly on expensive real-time PCR techniques employing labeled probes (Munthe-Fog et al. 2007; Ojurongbe et al. 2012), direct sequencing (Hummelshoj et al. 2005; Messias-Reason et al. 2009) or reverse-hybridization technology (Cedzynski et al. 2007; Haerynck et al. 2012). The procedures described in this report give clear results that are simple to analyze and interpret without costly equipment. They provide an opportunity to study four SNPs that influence the serum concentration of L-ficolin and/or its ligand binding capacity, and therefore are potentially important for clinical investigations. The results obtained with the use of this new cost-effective method appear to be entirely consistent with direct sequencing (100 % concordance).
References
Assaf A, Hoang TV, Faik I, Aebischer T, Kremsner PG, Kun JF, Velavan TP (2012) Genetic evidence of functional ficolin-2 haplotype as susceptibility factor in cutaneous leishmaniasis. PLoS One 7:e34113
Cedzynski M, Nuytinck L, Atkinson AP, St Swierzko A, Zeman K, Szemraj J, Szala A, Turner ML, Kilpatrick DC (2007) Extremes of L-ficolin concentration in children with recurrent infections are associated with single nucleotide polymorphisms in the FCN2 gene. Clin Exp Immunol 150:99–104
de Rooij BJ, van Hoek B, ten Hove WR, Roos A, Bouwman LH, Schaapherder AF, Porte RJ, Daha MR, van der Reijden JJ, Coenraad MJ, Ringers J, Baranski AG, Hepkema BG, Hommes DW, Verspaget HW (2010) Lectin complement pathway gene profile of donor and recipient determine the risk of bacterial infections after orthotopic liver transplantation. Hepatology 52:1100–1110
de Rooij BJ, van der Beek MT, van Hoek B, Vossen AC, Rogier Ten Hove W, Roos A, Schaapherder AF, Porte RJ, van der Reijden JJ, Coenraad MJ, Hommes DW, Verspaget HW (2011) Mannose-binding lectin and ficolin-2 gene polymorphisms predispose to cytomegalovirus (re)infection after orthotopic liver transplantation. J Hepatol 55:800–807
Endo Y, Sato Y, Matsushita M, Fujita T (1996) Cloning and characterization of the human lectin P35 gene and its related gene. Genomics 36:515–521
Haerynck F, Van Steen K, Cattaert T, Loeys B, Van Daele S, Schelstraete P, Claes K, Van Thielen M, De Canck I, Mahachie John JM, De Baets F (2012) Polymorphisms in the lectin pathway genes as a possible cause of early chronic Pseudomonas aeruginosa colonization in cystic fibrosis patients. Hum Immunol 73:1175–1183
Herpers BL, Immink MM, de Jong BA, van Velzen-Blad H, de Jongh BM, van Hannen EJ (2006) Coding and non-coding polymorphisms in the lectin pathway activator L-ficolin gene in 188 Dutch blood bank donors. Mol Immunol 43:851–855
Hoang TV, Toan NL, le Song H, Ouf EA, Bock CT, Kremsner PG, Kun JF, Velavan TP (2011) Ficolin-2 levels and FCN2 haplotypes influence hepatitis B infection outcome in Vietnamese patients. PLoS One 6:e28113
Hummelshoj T, Munthe-Fog L, Madsen HO, Fujita T, Matsushita M, Garred P (2005) Polymorphisms in the FCN2 gene determine serum variation and function of Ficolin-2. Hum Mol Genet 14:1651–1658
Hummelshoj T, Munthe-Fog L, Madsen HO, Garred P (2008) Functional SNPs in the human ficolin (FCN) genes reveal distinct geographical patterns. Mol Immunol 45:2508–2520
Kilpatrick DC, Chalmers JD (2012) Human L-ficolin (ficolin-2) and its clinical significance. J Biomed Biotechnol 2012:138797
Kilpatrick DC, Fujita T, Matsushita M (1999) P35, an opsonic lectin of the ficolin family, in human blood from neonates, normal adults, and recurrent miscarriage patients. Immunol Lett 67:109–112
Matsushita M (2010) Ficolins: complement-activating lectins involved in innate immunity. J Innate Immun 2:24–32
Matsushita M, Endo Y, Taira S, Sato Y, Fujita T, Ichikawa N, Nakata M, Mizuochi T (1996) A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin. J Biol Chem 271:2448–2454
Meijvis SC, Herpers BL, Endeman H, de Jong B, van Hannen E, van Velzen-Blad H, Krediet RT, Struijk DG, Biesma DH, Bos WJ (2011) Mannose-binding lectin (MBL2) and ficolin-2 (FCN2) polymorphisms in patients on peritoneal dialysis with staphylococcal peritonitis. Nephrol Dial Transplant 26:1042–1045
Messias-Reason IJ, Schafranski MD, Kremsner PG, Kun JF (2009) Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease. Clin Exp Immunol 157:395–399
Munthe-Fog L, Hummelshoj T, Hansen BE, Koch C, Madsen HO, Skjodt K, Garred P (2007) The impact of FCN2 polymorphisms and haplotypes on the ficolin-2 serum levels. Scand J Immunol 65:383–392
Ojurongbe O, Abou Ouf E, Van Tong H, Toan NL, Song LH, Luz PR, Messias-Reason IJ, Nurjadi D, Zanger P, Kun JF, Kremsner PG, Velavan TP (2012) Reliable and rapid characterization of functional FCN2 gene variants reveals diverse geographical patterns. BMC Med Genet 13:37
Ouf EA, Ojurongbe O, Akindele AA, Sina-Agbaje OR, Van Tong H, Adeyeba AO, Kremsner PG, Kun JF, Velavan T (2012) Ficolin-2 levels and FCN2 genetic polymorphisms as a susceptibility factor in schistosomiasis. J Infect Dis 206:562–570
Acknowledgments
This work was partially supported by Ministry of Science and Higher Education and National Science Centre (Poland), grants no N401 021035 and N402 353438. Authors are very grateful to Dr. David C. Kilpatrick for critical reading of the manuscript.
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The authors declare that they have no conflict of interest.
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Szala, A., St. Swierzko, A. & Cedzynski, M. Cost-effective procedures for genotyping of human FCN2 gene single nucleotide polymorphisms. Immunogenetics 65, 439–446 (2013). https://doi.org/10.1007/s00251-013-0696-7
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DOI: https://doi.org/10.1007/s00251-013-0696-7