Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease
Ficolins are pattern-recognition proteins involved in innate immunity, which upon binding to their specific pathogen-associated molecular patterns on the microbial surfaces trigger the immune response either by binding to collectin cellular receptors or by initiating the complement lectin pathway. In humans, three ficolin genes have been identified, which encode ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen). Ficolin-2 was shown to bind to lipoteichoic acid, a cell wall constituent in all Gram-positive bacteria such as Streptococcus pyogenes, which is the aetiological agent of rheumatic fever (RF) and its most severe sequelae, chronic rheumatic heart disease (CRHD). Here we investigated polymorphisms in the promoter region of the FCN2 gene (at positions −986/−602 and +4) in 122 patients with RF and CRHD and in 210 healthy subjects from the same geographic region and socioeconomic background. The haplotype −986/−602/−4 G/G/A, which is related to low levels of L-ficolin, was observed more frequently in the CRHD group when compared to the healthy subjects [99/162, 61·1% versus 211/420, 50·2%, odds ratio (OR) 1·6, confidence interval (CI) 95% 1·1–2·3, P = 0·021]. The haplotype −986/−602/−4 A/G/A was observed more frequently in the healthy group when compared to the affected (RF plus CRHD) subjects (31/420, 7·4% versus 6/244, 2·5%, OR 3·2, CI 95% 0·13–0·77, P = 0·008). Based on those findings, one can conclude that polymorphisms associated with low levels of L-ficolin level may predispose an individual to recurrent and/or more severe streptococcal infection.
Rheumatic fever (RF) is an autoimmune disease that results from a humoral and cellular altered response to an oropharyngeal infection with Streptococcus pyogenes. The disease affects 3–4% of genetically susceptible and untreated children and adolescents (aged 5–18 years). The clinical manifestations of RF include carditis, polyarthritis, chorea, erythema marginatum and/or subcutaneous nodules . Although arthritis is the most frequent manifestation of RF affecting 75% of the children, the most severe manifestation is carditis, which can cause permanent valvular damage in 30–45% of the affected children and young adults leading to chronic rheumatic heart disease (CRHD) . This chronic carditis is still the major cause of acquired cardiac valvulopathy affecting children and young adults throughout the world, being responsible for some 233 000 deaths annually . RF/CRHD are complex diseases with involvement of genetic as well as environmental factors in its pathogenesis . Several markers have been reported to be associated with RH/CRHD. While different human leucocyte antigen (HLA) alleles have been related to susceptibility to the disease in distinct populations, HLA-DR7 showed an association in different populations . Non-HLA genes, including MBL2[5,6] and tumour necrosis factor (TNF)-α genes , have also been shown to influence disease susceptibility. Both adaptive and innate responses, including the complement system, are believed to be involved in the pathogenesis of RF/RHD.
Ficolins are pattern-recognition proteins (PRPs) involved in the innate immune response. Ficolins bind to specific pathogen-associated molecular patterns (PAMPs) on microorganism surfaces, triggering the innate immune response by either binding to collectin cellular receptors or initiating the complement lectin pathway . The ficolins are synthesized as a single polypeptide containing N-collagen-like and C-terminal fibrinogen-like sugar binding domains, which are oligomerized into higher oligomeric forms . Both ficolin-2 and ficolin-3 are able to interact with the mannan-binding lectin (MBL)-associated serine proteases, promoting activation of the complement cascade . Other putative functions of ficolins include binding to late apoptotic cells, apoptotic bodies and necrotic cells enhancing their uptake by macrophages . Ficolin-2 was shown to bind to different clinically relevant bacteria, such as S. pyogenes and S. agalactiae[11,12]. Ficolin 2 binds specifically to lipoteichoic acid, a cell wall constituent of all Gram-positive bacteria, and thereby activates the lectin pathway of complement.
In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode, respectively, ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen) . The three different genes have divergent function, sequence and cell specificity. Single nucleotide polymorphisms (SNPs) in the promoter regions (−986, −602, −557, −64 and −4) of the FCN2 gene as well as in exons 3, 6 and 8 have been described recently [13,14]. FCN2 promoter polymorphisms −986, −602 and −4 are associated with changes in the ficolin-2 serum concentrations, whereas two polymorphisms located in exon 8, encoding the fibrinogen-like domain (+6359 and +6424), are associated with increased or decreased ability of carbohydrate binding [13,15,16]. It has been shown that the presence of the nucleotide adenine (A) at positions −986 and −602 is related to high ficolin-2 serum levels (A/A > A/G > G/G) as well as the nucleotide guanine (G) at the position −4 (G/G > G/A > G/G) . In previous studies, we reported an association between haplotypes/genotypes related to higher expression of serum levels of MBL, another constituent of the lectin pathway, with acute and chronic rheumatic carditis [5,6]. In this study, we assessed further the frequency of the diplotypes and haplotypes resulting from the SNPs located at positions −986, −602 and −4 of promoter region of the FCN2 gene in patients with history or RF.
Patients and controls
A total of 122 patients [37/30% males and 85/70% females, with a mean age of 38·31 ± 1·37 standard error (s.e.)/15·18 standard deviation (s.d.) years, 8–76 range] with a history of RF were included in the study. They were out-patients from the Children's Cardiologic Unit of the Hospital Pequeno Principe and from the Cardiology Out-patient Clinic at the University Hospital, Federal University of Paraná (UFPR), Curitiba, Southern Brazil. Rheumatic valvar disease was confirmed based on the clinical history as well as on transthoracic echocardiograms of all the patients. Forty-two patients presented no cardiac sequelae (RF) and 106 had a history of rheumatic mitral stenosis (CRHD). Among the patients with CRHD, 76 (71·69%) had undergone a previous invasive procedure due to rheumatic aetiology and 30 (28·30%) had not undergone any invasive procedure until the time of the study. Further clinical features of the patients with RF and CRHD are presented in Table 1. The control group included 210 healthy subjects from the same geographic region and socioeconomic background (with a mean age of 34·94 ± 0·81 s.e./9·95 s.d. years, 19–63 range), matched with the patients for age and ethnic background. The study was approved by the Ethics Committee of Human Research at the HC/UFPR and all subjects signed a free informed consent form.
Table 1. Clinical and demographical characteristics of the patients with rheumatic fever and rheumatic heart disease.
|Age (years)||22·63 ± 0·94 s.e. or 6·05 s.d.||46·24 ± 1·31 s.e. or 11·86 s.d.|
| Range||8 to 33||19 to 76|
|Sex|| || |
| Male||18 (44%)||19 (23·5%)|
| Female||23 (56%)||62 (76·5%)|
|Clinical characteristics|| || |
| Carditis||35 (85·3%)||81 (100%)|
| Arthritis||19 (46·3%)||n.a.|
| Subcutaneous nodules||4 (9·75%)||n.a.|
| Chorea||3 (7·31%)||n.a.|
| Erythema marginatum||1 (2·43%)||n.a.|
|Surgery|| || |
| Comissurotomy||no||7 (8·6%)|
| Balloon valvuloplasty||no||22 (27·1%)|
| Biological valve||no||21 (26%)|
| Metal valve||no||8 (9·9%)|
Genotyping promoter region of FCN2
DNA extraction was performed using QIAamp™ DNA extraction kits following the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). The promoter was amplified by polymerase chain reaction (PCR) from genomic DNA in a 1278 base pairs (bp) fragment, which also included exon 1. The primer sequences for promoter and exon1 region were forward 5′-ATT GAA GGA AAA TCC GAT GGG-3′, adopted from Hummelshoj et al., and reverse 5′-GAA GCC ACC AAT CAC GAA G-3′ (own sequence). In total, 100 ng of genomic DNA was amplified in a 25 µl volume of reaction mixture containing 1× reaction buffer (20 mM Tris pH 8·8, 10 mM KCl, 1·5 mM MgCl2 and 0·1% Triton X-100), 1× Q-solution (Qiagen), 0·2 mM 2′-deoxynucleoside 5′-triphosphate (dNTP), 0·5 mM MgCl2, 0·5 µM of each primer and 1·0 U Taq polymerase (Qiagen). The cycling conditions used for amplification were 95°C for 5 min; 30 cycles of 95°C for 40 s, 59°C for 60 s, 72°C for 90 s, and finished with 72°C for 3 min and 15°C for 10 min. The amplified polymerase chain reaction (PCR) fragments were visualized on a 1% (w/v) agarose gel electrophoresis after staining with SYBR green I (Biozym Diagnostik GmbH, Wien, Austria). Prior to DNA sequencing the fragments were purified using the EZNA Cycle-Pure kit following the manufacturer's instructions (PeqLab Biotechnologie GmbH, Erlangen, Germany). Sequencing was performed with the BigDye® Terminator version 1·1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Separation of strands were analysed on an automated sequencer (ABI Prism 3100 Genetic Analyzer; Applied Biosystems). FCN2 genotypes and haplotypes were identified using the software SeqScape version 2·5 (Applied Biosystems).
Independence between cases and controls was tested using the two-tailed Fisher's exact test. A backward logistic regression model was also applied to assess the association between RHD and SNPs diplotypes at positions −986, −602 and −4. Statistical analyses were performed using MedCalc (MedCalc Software, Mariakerke, Belgium). A P-value less than 0·05 was considered to be significant. Hardy–Weinberg equilibrium was calculated using arlequin software version 3·11 ( http://cmpg.unibe.ch/software/arlequin3/). Data are presented as mean ± s.e./s.d.
Clinical characteristics of the RF and CRHD patients investigated are shown in Table 1. FCN2 diplotypes and haplotypes frequencies were in accordance with Hardy–Weinberg expectations for both groups
The haplotype −986/−602/−4 A/G/A was observed less frequently in the affected (RF plus CRHD) and in the RF subjects when comparing with the healthy group [6/244, 2·5% versus 31/420, 7·4% odds ratio (OR) 0·32, 95% confidence interval (CI) 0·13–0·77, P = 0·008 and 1/82, 1·2% versus 31/420, 7·4%, OR 0·15, 95% CI 0·02–1·15, P = 0·044, respectively), Table 2. On the other hand, the frequency of the haplotype 986/−602/−4 G/G/A was increased in CRHD when compared to the healthy subjects (99/162, 61·1% versus 211/420, 50·2%, OR 1·6, 95% CI 1·08–2·25, P = 0·021). This haplotype (986/−602/−4 G/G/A) also showed a higher frequency in the CRHD patients in comparison with the RF patients, but with no statistical significance (99/162, 61·1% versus 42/82, 51·2%, P = 0·170). In addition, a higher frequency of the haplotype −986/−602/−4 A/G/G was observed in the RF group when compared to the RHD group and to the controls (25/82, 30·5% versus 26/162, 16%, OR 2·3, 95% CI 1·22–4·31, P = 0·012 and 25/82, 30·5% versus 85/335, 20·2%, OR 1·73, 95% CI 1·03–2·20, P = 0·057, respectively), Table 2. In addition, by using logistic regression, low- and high-producing FCN2 diplotypes at positions −986, −602 and −4 were analysed as risk factors for the development of CRHD. After adjusting for age, an OR of 1·78 (95% CI 0·89–3·55, P = 0·099) was observed for the low-producing FCN2 diplotype at position −986 (G/G). The haplotypes −986/−602/−4 A/A/G and G/A/A were observed only in the control subjects. There was no statistically significant difference in the distribution of FCN2 diplotypes or alleles on their own among RF or CRHD patients or controls (data not shown).
Table 2. FCN2 promoter haplotypes frequencies at positions −986/−602/−4 in patients with rheumatic fever and rheumatic heart disease.
|AGG||85||20·2||26|| 16·0 ||25|| 30·5 ||51||20·9||0·44(0·23–0·82)||0·012†|
| GGA ||211|| 50·2 ||99|| 61·1 ||42||51·2||141||57·8||1·56(1·10–2·30)||0·021‡|
|AGA||31|| 7·4 ||5||3·1||1|| 1·2 ||6|| 2·5 ||0·32(0·13–0·77)||0·008§|
| GG G||6||1·4||1||0·6||2||2·4||3||1·2||n.a.||n.s.|
| G AA||5||1·2||0||0||0||0||0||0||n.a.||n.s.|
|Chromosomes||420|| ||162|| ||82|| ||244|| || || |
To the best of our knowledge, this is the first study addressing FCN2 gene polymorphisms in patients with RF or CRHD. Ficolins are molecules involved in the innate immune response, thus having an important role in the first line of defence against pathogens. They are able to bind to specific PAMPs expressed on the surface of microorganisms and trigger the complement cascade through interactions with mannose-binding lectin-associated serine proteases (MASPs). Moreover, upon binding to collectin receptors, ficolins were shown to activate immune cells to secrete cytokines such as TNF-α, interleukin (IL)-1 and IL-8 . Previously, low ficolin-2 serum levels have been related to recurrent respiratory infections in children  and FCN2 functional haplotypes have been associated with the development of clinical leprosy . It has also been reported that binding properties of ficolin-2 differed from MBL and ficolin-3 , suggesting that the binding of each lectin is directed towards a specific and different PAMP. Also of importance is the finding that ficolin-2, but not MBL or ficolin-3 binds specifically to lipoteichoic acid, which is the most common antigen expressed on Gram-positive bacterial cells . In fact, ficolin-2/MASP complexes were shown to bind to different Gram-positive bacteria including S. pyogenes, thereby initiating complement activation . Thus, the repertoire of pathogens recognized by ficolin-2 enlarges that recognized by MBL and the specificity of innate response.
According to clinical, epidemiological and immunological data, S. pyogenes is considered as the aetiological agent of RF . It is known that repeated and/or non-treated oropharyngeal streptococcal infections are necessary to trigger the autoimmune responses that lead ultimately to the development of RF and CRHD in susceptible individuals . In this study, a significant higher frequency of the haplotype −986/−602/−4 G/G/A, which is associated with low ficolin-2 serum levels, was observed in patients with CRHD when compared to the healthy subjects and, to a lesser extent, to RF. Interestingly, when analysing the affected group as a whole or RF only no significant differences were observed regarding the same haplotype. This result suggests that this particular haplotype may play a role in the progression of RF to its chronic form. As repeated S. pyogenes infections are needed for the development of RHD, low serum levels of L-ficolin may be related to an impaired clearance of the pathogen, predisposing the individual to the chronic form of this autoimmune disease. In contrast, −986/−602/−4 A/G/A showed a protective effect against RF (OR 0·15) and against the development of both diseases (OR 0·23). The haplotype −986/−602/−4 A/G/G was also associated with protection to CRHD when comparing to RF (OR = 0·44).
In previous studies, elevated serum levels of MBL as well as high-producing MBL2 genotypes were shown to increase the risk of acute and chronic rheumatic carditis in patients with history of RF [6,21], suggesting a proinflammatory role for MBL in the disease. In contrast, no association of MASP-2 deficiency due to homozygous Asp120Gly mutation (120) was observed in the same cohort of patients . Similarly to ficolins, MBL is also able to activate complement through interactions with MASPs . MBL is capable of binding to N-acetyl-D-glucosamine, a molecule that is also present on human heart valves . It seems, therefore, that the proteins of the lectin pathway, MBL, MASP-2 and ficolin-2, have a distinct role in the pathogenesis of RF/CRHD. Whereas low levels of ficolin-2 due to a mutation in the transcriptional level might predispose the individual to recurrent and/or more severe streptococcal infection, activation of the complement system due to high levels of MBL may be involved in the inflammatory process which leads to cardiac injury in RF/CRHD patients.
Finally, these data suggest that FCN2 promoter haplotypes are associated with the susceptibility of RF and its chronic form CRHD, with −986/−602/−4 G/G/A haplotype representing a novel risk factor and A/G/G and A/G/A being protective against the development of CRHD, a disorder whose pathogenesis is still not well understood.
The authors have no conflicts of interest to declare.
This study received funds from the European Commission through a grant to J. F. J. K. (TRANCHI).