Acute rheumatic fever is the most important immune-mediated complication of upper respiratory tract infection with group A, β-haemolytic streptococci. The immune mechanisms that cause concomitant inflammation of synovial joints and cardiac valves are poorly defined. Acute rheumatic fever shows a wide spectrum of clinical features including carditis, arthritis, chorea, subcutaneous nodules, and erythema marginatum. Rheumatic heart disease is one of the most severe manifestations of acute rheumatic fever.Reference Carapetis, McDonald and Wilson 1
Although the pathogenesis of the disease is not fully understood, it has been considered to occur as a result of abnormal immune response of the susceptible host to specific bacterial agents.Reference Carapetis, McDonald and Wilson 1 Antigenic similarity between group A streptococcal antigens and cardiac, articular, and central nervous system proteins has been proposed as the triggering factor leading the autoimmunity in susceptible individuals with genetic predisposition.Reference Guilherme, Ramasawmy and Kalil 2
Mannose-binding lectin-2 and macrophage migration inhibitory factor play important roles in the pathogenesis of several acute and chronic inflammatory/autoimmune disorders.Reference Ruskamp, Hoekstra, Rovers, Schilder and Sanders 3 , Reference Renner, Roger and Calandra 4 Mannose-binding lectin-2 is an acute phase inflammatory protein involved in the primary defense against microorganisms. Circulating mannose-binding lectin-2 binds to the sugars on the surface of a variety of pathogens, including group A streptococcus.Reference Turner 5 It is encoded by the mannose-binding lectin-2 gene, and variations of the gene correlate with the insufficiency of mannose-binding lectin-2 in the plasma. Recently, it has been shown that there may be an association between rheumatic heart disease and mannose-binding lectin-2 gene polymorphisms.Reference Messias Reason, Schafranski, Jensenius and Steffensen 6 – Reference Schafranski, Ferrari, Scherner, Torres, Jensenius and Messias Reason 8 Macrophage migration inhibitory factor is expressed in several types of cells and tissue, including monocytes/macrophages, and plays a central role in the host response to endotoxinaemia.Reference Calandra, Spiegel, Metz and Bucala 9 Variations of the macrophage migration inhibitory factor gene have been associated with the susceptibility or severity of chronic systemic inflammatory diseases such as juvenile idiopathic rheumatoid arthritis, glomerulonephritis, ulcerative colitis, atopy, or sarcoidosis.Reference Renner, Roger and Calandra 4 The possible role of macrophage migration inhibitory factor in children with acute rheumatic fever has not yet been investigated.
The aim of the present study is to investigate any possible association between mannose-binding lectin-2 and macrophage migration inhibitory factor gene polymorphisms and acute rheumatic fever in children and to investigate the association between the identified genotypes/alleles and their clinical features.
Material and methods
Study population
In this study, 38 unrelated children with acute rheumatic fever and 40 age- and sex-matched healthy controls were investigated. All of the patients fulfilled the Jones criteria for the diagnosis of acute rheumatic fever.Reference Carapetis, McDonald and Wilson 1 The medical records of all children with acute rheumatic fever were reviewed. The children were diagnosed with acute rheumatic fever in the Pediatric Cardiology Clinic of Gaziantep University Hospital and all of them were evaluated by the same paediatric cardiologist. We confirmed that they met the Jones criteria from their medical records. In all, 14 patients (36.8%) had a previous history of acute rheumatic fever. The other 24 children (63.2%) entered into the study with a new diagnosis of acute rheumatic fever. Peripheral blood samples for anti-streptolysin O, C-reactive protein, and erythrocyte sedimentation rate levels were obtained at the time of diagnosis of acute rheumatic fever.
The study was approved by the local ethics committee, and informed consent was obtained from the patients and/or their parents.
Deoxyribonucleic acid isolation
Genomic deoxyribonucleic acid was extracted from peripheral blood samples using the salting out procedure.Reference Miller, Dykes and Polesky 10
Genotyping
Genotyping of mannose-binding lectin-2 gene (codon 54 A/B)
Polymerase chain reaction was performed using forward (5′-TAGGACAGAGGGCATGCTC-3′) and reverse (5′-CAGGCAGTTTCCTCTGGAAGG-3′) primers in a 25-microliter volume containing 50-nanogram deoxyribonucleic acid, 2-millimolar deoxyribonucleotide triphosphates, 2 nanomols of each primer, 1.5-millimolar MgCl2 and 3 unit Taq polymerase. The PCR products which were digested with BanI restriction enzyme, were 349 base pairs. BanI digestion was performed at 50°C for 60 minutes with 5 unit enzyme. After enzyme digestion, products were visualised by electrophoresis on 3% agarose gel. The BanI restriction site is present on wild-type allele A and absent on variant allele B.Reference Vardar, Pehlivan and Onay 11
Genotyping of macrophage migration inhibitory factor gene (−173 G/C)
Polymerase chain reaction was performed using forward (5′-ACTAAGAAAGACCCGAGGC-3′) and reverse (5′-GGGGCACGTTGGTGTTTAC-3′) primers. For migration inhibitory factor (−173), a 330-base pair fragment was amplified, which was then digested with AluI restriction enzyme overnight at 37°C. The products were then separated on 3% agarose gel. The product contains two restriction sites for allele C and one of these sites is destroyed when allele G is present.Reference Akcali, Pehlivan, Pehlivan, Sever and Neyal 12
Statistical analysis
All statistical analyses were performed with the Statistical Package for the Social Science for Windows (version 18.0; SPSS Inc., Chicago, Illinois, United States of America). The results are given as mean plus or minus standard deviation, whereas allele frequencies and the distribution of genotype are given as percent. The clinical features and mannose-binding lectin-2/macrophage migration inhibitory factor gene polymorphisms were compared using the chi-square and the Fisher exact tests. Statistical significance was considered a p-value less than 0.05. The Hardy–Weinberg equilibrium was calculated using the de Finetti program. 13
Results
Clinical features
The age ranged between 5 and 15 years, with a mean of 10.71 plus or minus 2.51 years, in children with acute rheumatic fever (n is equal to 38, 17 girls and 21 boys) and between 4 and 17 years, with a mean of 10.37 plus or minus 3.81, in healthy controls (n is equal to 40, 16 girls and 24 boys). In all, 23 (60.5%) patients had a history of antecedent upper respiratory tract infection. The other clinical features of the patients are summarised in Table 1.
Table 1 Clinical features of the children with acute rheumatic fever.
Genotype and allele frequencies of the genes
The distribution of AA, AB, and BB genotypes for mannose-binding lectin-2 was 55.3%, 34.2%, and 10.5%, respectively, in children with acute rheumatic fever compared with 65%, 35%, and 0%, respectively, in controls. BB genotype was significantly higher in the patient group (Table 2). The allele frequency of A/B in mannose-binding lectin-2 was 72.4% and 27.6% in children with acute rheumatic fever compared with 82.5% and 17.5% in controls (Table 2). The distribution of GG, GC, and CC genotypes for macrophage migration inhibitory factor was 47.4%, 31.6%, and 21% in children with acute rheumatic fever compared with 57.5%, 40%, and 2.5% in controls. CC genotype was significantly higher in the patient group (Table 3). The allele frequency of G/C in macrophage migration inhibitory factor was 63.2% and 36.8% in children with acute rheumatic fever compared with 77.5% and 22.5% in controls. C allele was higher in the patient group (Table 3). The observed genotype counts were deviated significantly from those expected according to the Hardy–Weinberg equilibrium for macrophage migration inhibitory factor gene polymorphism (p-value is equal to 0.047).
Table 2 Genotype and allele frequencies of MBL2 gene (codon 54) polymorphism in children with ARF and controls.
ARF = acute rheumatic fever; HWE = Hardy–Weinberg equilibrium; MBL2 = mannose-binding lectin-2
Table 3 Genotype and allele frequencies of macrophage MIF gene (−173 G/C) polymorphism in children with ARF and controls.
ARF = acute rheumatic fever; HWE = Hardy–Weinberg equilibrium; MIF = migration inhibitory factor
Association between the identified genotypes and patients clinical/laboratory features: we investigated the correlations of mannose-binding lectin-2/macrophage migration inhibitory factor genotypes with clinical and laboratory findings of acute rheumatic fever such as carditis, polyarthritis, fever, C-reactive protein level, erythrocyte sedimentation rate, and increased streptococcal antibody titre.
All of the patients with chorea had AA genotype of mannose-binding lectin-2 gene (p-value is equal to 0.031). GG genotype of macrophage migration inhibitory factor gene was higher in patients with a history of antecedent upper respiratory tract infection (p-value is equal to 0.013).
We found no relationship between mannose-binding lectin-2/macrophage migration inhibitory factor genotypes and other clinical or laboratory parameters (data were not shown).
Discussion
The pathogenesis of acute rheumatic fever is complicated and environmental/genetic factors contribute to its aetiology, and the presence of different clinical features may also show wide genetic heterogeneity. Acute rheumatic fever is clearly the result of an exaggerated immune response to specific bacterial agents in a susceptible host, and innate/adaptive immune responses are associated with the development of acute rheumatic fever.Reference Carapetis, McDonald and Wilson 1 , Reference Guilherme and Kalil 14 It has been shown that mannose-binding lectin-2 and macrophage migration inhibitory factor play an important role in immune response.Reference Froidevaux, Roger, Martin, Glauser and Calandra 15 , Reference Worthley, Bardy and Mullighan 16 We thought that searching the relationship between acute rheumatic fever and mannose-binding lectin-2/macrophage migration inhibitory factor gene polymorphisms may be helpful in understanding the pathogenesis of the disease.
Serum mannose-binding lectin-2 concentrations vary widely from person to person because of the variant alleles (B, C, and D, denoting the substitution of aspartic acid for glycine at codon 54, the substitution of glutamic acid for glycine at codon 57, and the substitution of cysteine for arginine at codon 52, respectively) in exon 1 of the mannose-binding lectin-2 gene.Reference Takahashi, Ip, Michelow and Ezekowitz 17 Each of the three structural variants influences the stability of the final protein product, resulting in reduced serum levels.Reference Garred, Larsen, Madsen and Koch 18 Mannose-binding lectin deficiency is associated with increased risk of infectious and autoimmune diseases.Reference Worthley, Bardy and Mullighan 16
In this study, we found that homozygous subjects for the variant allele of codon 54 of mannose-binding lectin-2 gene is a risk factor for acute rheumatic fever in children. Our findings are consistent with those reported by Ramasawmy et al, who suggested that mannose-binding lectin-2 deficiency may be a contributing pathogenic factor in Brazilian patients with rheumatic heart disease. They found that homozygous or compound heterozygous defective alleles in exon 1 of the mannose-binding lectin-2 gene are associated with chronic severe aortic regurgitation of rheumatic aetiology.Reference Ramasawmy, Spina and Fae 7 Schafranski et alReference Schafranski, Ferrari, Scherner, Torres, Jensenius and Messias Reason 8 reported that the frequency of variant alleles was significantly decreased in patients with a history of acute and chronic carditis, and Messias-Reason et alReference Messias Reason, Schafranski, Jensenius and Steffensen 6 demonstrated that there is a lower frequency of the variant alleles in patients with rheumatic heart disease. The reason for this variety is unclear, but it could show the multifactorial aetiology of acute rheumatic fever. Mannose binding lectin-mediated clearance of infectious agents provokes a pro-inflammatory response, while mannose binding lectin-dependent clearance of apoptotic cells promotes an anti-inflammatory response.Reference Takahashi, Ip, Michelow and Ezekowitz 17 It is most probable that the defective mannose-binding lectin-2 allele is one of several other predisposing risk factors in the development of acute rheumatic fever. It is likely to act in synergy with other genetic and environmental factors.Reference Ramasawmy, Spina and Fae 7 These findings have led to the idea that recombinant human mannose-binding lectin-2 therapy might be used as prophylaxis for infectious disease in patients with low-producing mannose-binding lectin-2 genotypes.Reference Takahashi, Ip, Michelow and Ezekowitz 17
We evaluated whether the presence of variant mannose-binding lectin-2 alleles would be associated with clinical and laboratory findings of acute rheumatic fever. Disease-related clinical or laboratory parameters did not show any significant association with mannose-binding lectin-2 genotype, except for chorea. Children with AA genotype (wild type) tended to have chorea compared with children with BB genotype (variant). Sydenham chorea is usually a delayed and isolated manifestation of acute rheumatic fever.Reference Carapetis, McDonald and Wilson 1 The main theory for the development of chorea in acute rheumatic fever is an autoimmune reaction resulting from antigenic mimicry between group A streptococcal antigens and cardiac, articular, and central nervous system proteins.Reference Guilherme and Kalil 14 The immunopathogenesis of chorea is thought to involve streptococcus-induced antibodies that cross-react with antigens of the basal ganglia.Reference Dale 19 Garred et alReference Garred, Madsen and Marquarth 20 reported that patients with late-onset rheumatoid arthritis who were also homozygous for wild-type alleles in exon 1 of mannose-binding lectin-2 gene were more likely to show evidence of persistent inflammation. We hypothesised that persistent inflammation due to the presence of wild type in these children may create a predisposition to chorea. However, this hypothesis needs to be clarified by further, detailed studies.
Macrophage migration inhibitory factor plays an important role in the control of inflammation and innate immune response to microbial invasion Mutations in the human macrophage migration inhibitory factor gene would predispose affected hosts to altered susceptibility to or severity of inflammatory or infectious disease. Four polymorphisms of the human macrophage migration inhibitory factor gene (−2794, −2173, +1254, +1656) have been reported.Reference Renner, Roger and Calandra 4 Patients with −173C allele, that is, guanine-to-cytosine transition at position −173, had increased levels of macrophage migration inhibitory factor, and increased macrophage migration inhibitory factor concentrations have been associated with severe clinical manifestations, high severity scores, and poor outcome of inflammatory disease.Reference Renner, Roger and Calandra 4 In this study, we found that macrophage migration inhibitory factor −173C allele is more prevalent in patients with acute rheumatic fever than in controls. Berdeli et alReference Berdeli, Özyürek and Ülger 21 from Turkey reported that allele and genotype distributions of macrophage migration inhibitory factor gene −173G/C polymorphism did not differ between children with juvenile rheumatoid arthritis and the control group. However, it was previously reported that macrophage migration inhibitory factor −173C allele was associated with juvenile idiopathic arthritis.Reference Donn, Alourfi and De Benedetti 22
No association was found between macrophage migration inhibitory factor genotypes and clinical or laboratory parameters of acute rheumatic fever in our study. However, the antecedent upper respiratory tract infection history was more common in GG homozygous patients. The polymorphism of macrophage migration inhibitory factor −173C allele causes increased plasma levels of macrophage migration inhibitory factor.Reference Renner, Roger and Calandra 4 Increased macrophage migration inhibitory factor concentrations may predispose the host to more severe immune reactions and loss of function of macrophage migration inhibitory factor gene may affect the innate immune response of the host.Reference Renner, Roger and Calandra 4 These findings may partially explain the presence of a history of antecedent upper respiratory tract infection in patients with acute rheumatic fever with GG genotype and also suggest that macrophage migration inhibitory factor may be an important promising candidate for molecular preventive therapy for acute rheumatic fever and usage of anti-macrophage migration inhibitory factor therapy in the prophylaxis of some infectious diseases.Reference Calandra, Spiegel, Metz and Bucala 9 , Reference Froidevaux, Roger, Martin, Glauser and Calandra 15
The major limitation of this study is the small number of included patients with acute rheumatic fever. However, despite this limitation we found significant results for mannose-binding lectin-2 and macrophage migration inhibitory factor gene polymorphisms. Furthermore, the present study is the first to investigate the macrophage migration inhibitory factor gene polymorphism in children with acute rheumatic fever.
In conclusion, acute rheumatic fever is a complex multifactorial disease. Mannose-binding lectin-2 and macrophage migration inhibitory factor are candidate genes for the investigation of the pathogenesis of acute rheumatic fever. Our study suggests that BB genotype polymorphism of mannose-binding lectin-2 (codon 54) and carriage of macrophage migration inhibitory factor −173C allele may contribute to the development of acute rheumatic fever in children. However, further studies in a larger population are needed to confirm the results.
