Human Immunology 75 (2014) 1197–1202
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Association of MASP2 polymorphisms and protein levels with rheumatic fever and rheumatic heart disease Sandra Jeremias dos Santos Catarino a, Angelica Beate Winter Boldt a,b, Marcia Holsbach Beltrame a, Renato Mitsunori Nisihara a, Marcelo Derbli Schafranski c, Iara Jose de Messias-Reason a,⇑ a b c
Laboratório de Imunopatologia Molecular, Departamento de Patologia Médica, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, Brazil Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brazil Departamento de Medicina, Universidade Estadual de Ponta Grossa, Paraná, Brazil
a r t i c l e
i n f o
Article history: Received 21 March 2014 Accepted 6 October 2014 Available online 12 October 2014 Keywords: Rheumatic fever Rheumatic heart disease MASP2 Polymorphism Haplotype-specific genotyping
a b s t r a c t MASP-2 is a key protein of the lectin pathway of complement system. Several MASP2 polymorphisms were associated with MASP-2 serum levels or functional activity. Here we investigated a possible association between MASP2 polymorphisms and MASP-2 serum levels with the susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD). We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 145 patients with history of RF from south Brazil (103 with RHD and 42 without cardiac lesion [RFo]) and 342 healthy controls. MASP-2 levels were determined by ELISA. The low MASP-2 producing p.377A and p.439H variants were negatively associated with RF (P = 0.02, OR = 0.36) and RHD (P = 0.01, OR = 0.25). In contrast, haplotypes that share the intron 9 – exon 12 g.1961795C, p.371D, p.377V and p.439R polymorphisms increased the susceptibility to RHD (P = 0.02, OR = 4.9). MASP-2 levels were associated with MASP2 haplotypes and were lower in patients (P < 0.0001), which may reflect protein consumption due to complement activation. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatic fever (RF) and its most severe sequel chronic rheumatic heart disease (RHD) are chronic inflammations that follow oropharynx infection by b-hemolytic Streptococcus group A. The disease occurs in genetically predisposed children and teenagers (aged 3–19 years) affecting the heart, joints, nervous system and skin [1]. The onset of RF usually occurs two to three weeks following the initial pharyngitis, but in some cases the onset may be months later [2]. Carditis is the most severe clinical manifestation of RF, affecting about 30–50% of patients 4–8 weeks after the first RF episode [3]. In general, carditis progress to RHD, associated with chronic inflammation and stenosis of valve tissue, leading to permanent heart damage. RHD affects young adults and remains a
Abbreviations: MASP-2, mannan-binding lectin-associated serine protease 2; MASP2, MASP-2 gene; MBL, mannan-binding lectin; RF, rheumatic fever; RHD, rheumatic heart disease. ⇑ Corresponding author at: Laboratório de Imunopatologia Molecular, Serviço de Anatomia Patológica, Hospital de Clínicas, Federal University of Paraná (UFPR), R. General Carneiro, 181, CEP 80060-900 Curitiba, PR, Brazil. E-mail address: [email protected] (I.J. de Messias-Reason).
major public health problem in Brazil and other developing countries, due to high morbidity and mortality. RF incidence exceeds 50 per 100,000 children in some developing countries [4] and RHD global prevalence varies between 15 and 20 million cases [5]. About two million cases require repeated hospitalization and one million may need a heart transplant in 5–20 years, generating high costs to the health system [1,5,6]. In Brazil, the estimated rate is 10 million cases of streptococcal pharyngitis each year, resulting in 30,000 new cases of RF, of which approximately 15,000 progress to RHD [7]. The complement system serves as the backbone of innate immunity and supports the adaptive immune system in gaining momentum to respond. At present, more than 40 components of complement have been described [8]. The complement system is activated through the classical, alternative and lectin pathways. Complement activation leads to recruitment of inflammatory mediators, pathogen destruction and clearance of immune complexes and apoptotic cells [9,10]. The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrates or acetylated residues on the surface of pathogens, respectively [11]. MBL and ficolins are associated with serine
http://dx.doi.org/10.1016/j.humimm.2014.10.003 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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proteases named MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which mediate the formation of C3 convertase [11]. MASP-2 and MAp19 (also known as sMAP or small MAP) are encoded by the MASP2 gene, located on 1p36.23-31. MAp19 is a truncated protein resulting from alternative splicing and inclusion of exon 5 in the mature mRNA [12]. MASP-2 and MAp19 share two domains encoded by the first four exons (CUB1 – C1r/C1s, Uegf and bone morphogenetic protein-1 – and an epidermal growth factor (EGF)-like domain). Exon 5 encodes the four last amino acids exclusive of MAp19. Exons 6–11 encode a second CUB domain (CUB2) and two contiguous complement control protein modules (CCP1 and CCP2). Exon 12 encodes the activating peptide and serine protease domain [13]. MASP-2 plays a key role in the activation of the lectin pathway initiated by ficolins, colectin 11 or MBL. MAp19 binds MBL, but its function remains speculative [14]. MASP-2 deficiency was first described in a patient with multiple infections and autoimmune manifestations, due to an exon 3 mutation causing the exchange of aspartic acid with glycine at position 120 (p.D120G) [15]. Several other MASP2 polymorphisms, including g.1945560C>A in the promoter region, p.R99Q and p.P126L in exon 3, g.7164A>G in intron 4, g.7441G>A in intron 5, g.1961795C>T in intron 9, p.D371Y and p.V377A in exon 10, p.R439H and g.24762C>T in exon 12, were found associated with serum levels or functional activity of MASP-2 [16–19]. Some of them (p.126L, p.377A, p.439H – associated with low MASP-2 levels and p.371D – associated with high MASP-2 levels probably due to linkage disequilibrium with intronic variants) were associated with susceptibility to diseases: leprosy [19], hepatitis C [20], malaria [21], bacterial infections after orthotopic liver transplantation [22], Chagas disease [23] and rheumatoid arthritis [24]. These SNPs are distributed in ten main haplotypes comprised of four variant blocks. The first block contains the promoter variant at nucleotide position 1945560 and three amino acid variants at codons 99, 120 and 126 (ARDP, ARGP, CRDP, CQDP, CRDL); the second block contains the intron 4 and 5 variants (AG, GA, GG), the third block by the intron 9 and two amino acid variants at codons 371 and 377 (CDA, CDV, TDV), fourth block contains one amino acid variant at codon 439 and a synonymous variant at nucleotide position 24762 (RC, HC, RT). For practical reasons, the haplotypes were named according to their phylogenetic relationships. Five haplotypes belong to clade 1 and share g.24762C in exon 12. Among them, ⁄1A represents the most ancient haplotype, ⁄1B1-h and ⁄ 1B2-h share g.1961795T in intron 9 and are associated with higher MASP-2 levels (reason for the ‘‘h’’ suffix), ⁄1C1-l and ⁄1C2-l share p.126L and are associated with low MASP-2 levels (reason for the ‘‘l’’ suffix). Importantly, ⁄1C2-l also bears the deficiency-causing p.439H variant. Five other haplotypes belong to clade 2 and present g.24762T in exon 12: ⁄2A1, ⁄2A2-l (with p.377A), ⁄2B1-i and ⁄ 2B2A-i (both associated with intermediate – ‘‘i’’ – MASP-2 levels), and the MASP-2 deficiency-causing ⁄2B2B-l haplotype (with the p.120G variant) [18]. Variants in introns 4 and 5 mainly occur as two combinations in cis, AG and GA, in the ⁄1A, ⁄1B1-h, ⁄2A1, ⁄ 2B1-I and ⁄2B2A-I haplotypes, reason for which they are added between clasps after the haplotype name (e.g. ⁄1A [AG]). These polymorphisms probably modulate alternative exon 5 splicing, since GA is associated with higher MASP-2 and lower MAp19 levels, the opposite being true for AG [19].
2. Material and methods 2.1. Subjects and samples This study was approved by the local ethics committee (CEP/HC 2658.265/2011-11). We investigated a total of 145 patients with a
history of RF with mean age of 39 years (range = 18–89 years). All patients had a history of RF and were diagnosed according to Jones’ modified criteria [25]. Among them, 103 (71%) had RHD, confirmed by transthoracic echocardiogram showing rheumatic involvement of the mitral valve; 42 patients (29%) did not present RHD but had history of RF and were designated as ‘‘rheumatic fever only’’ (RFo) patients. Clinical characterization of RFo and RHD patients was described in a previous study [26]. None of the patients presented other inflammatory disease, neoplasia, infective endocarditis or other infection at the time of blood collection. The control group included 286 blood donors and 56 health workers and other volunteers (without history of rheumatic fever) from the same geographic region, with a mean age of 41.3 years (range = 18–61 years). Among the blood donors, 65 were from Hospital de Clínicas of the UFPR, 174 were from Centro de Hemoterapia e Hematologia do Paraná (Hemepar) and 47 from Biobanco of the Hospital Evangélico, paired with the patients according to age and ancestry. Differences in sex distribution between patients and controls and in age distribution between RHD and RFo patients were corrected with multivariate logistic regression (Table 1). 2.2. MASP2 genotyping A total of eleven MASP2 single nucleotide polymorphisms (SNPs) were investigated. Taking NG007289.1 as reference sequence, they were: g.4847A>C in the promoter (rs7548659), g.5557G>A (rs61735600), g.5620A>G (rs72550870) and g.5638C>T (rs56392418) in exon 3 (causing amino acid substitutions p.R99Q, p.D120G and p.P126L in the CUB1 domain, respectively), g.7164A>G (rs2273344) in intron 4, g.7441G>A (rs9430347) in intron 5, g.21081C>T in intron 9 (rs17409276), g.21370G>T (rs12711521) and g.21389T>C (rs2273346) in exon 10 (causing amino acid substitutions p.Y371D and p.V377A in the CCP2 domain, respectively) and g.24599G>A (rs12085877) and g.24762T>C (rs1782455) in exon 12 (encoding one non synonymous – p.R439H and one synonymous variant – p.S493=). Minor intronic alleles (g.7164G, g.7441A and g.21081T) have been formerly associated with MASP-2 levels higher than 600 ng/ml [18,19], whereas minor non synonymous alleles encoding p.120G, p.126L, p.377A and p.439H were associated with MASP-2 levels lower than 200 ng/ml. Among them, homozygotes for p.120G or p.439H are unable to activate the lectin pathway of complement [16,17]. The SNPs were identified by a multiplex sequence-specific amplification method (multiplex PCR-SSP), as previously described [18,19]. 2.3. MASP-2 assay MASP-2 concentrations were measured in the sera of 145 patients (42 RFo and 103 RHD) and 196 controls using enzymelinked immunosorbent assay (HK326, Hycult Biotechnology, Uden, The Netherlands). Both groups were homogeneous regarding MASP2 genotype distribution. Minimum concentration which can be measured is 1.6 ng/ml. Color intensity was evaluated at 450 nm in an ELISA reader. 2.4. Statistics Genotype, allele and haplotype frequencies were obtained by direct counting. SNPs distributed from the promoter to exon 12 were phased with the SSP primers. In most cases, the phase between distantly situated SNPs could be deduced due to strong linkage disequilibrium between the variants [18]. Hardy– Weinberg equilibrium and homogeneity between genotype distributions were tested using ARLEQUIN software package version 3.5.1.3 [27]. MASP-2 levels were tested for normality
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S.J.d.S. Catarino et al. / Human Immunology 75 (2014) 1197–1202 Table 1 Demographic characteristics of RF and RHD patients.
n Male (%) Female (%) Euro-Brazilians (%) Afro-Brazilians (%) Mean age ± SD Mean age female ± SD Mean age male ± SD
n Male (%) Female (%) Euro-Brazilians (%) Afro-Brazilians (%) Mean age ± SD Mean age female ± SD Mean age male ± SD
Patients
Controls
145 43 (30%) 102 (70%) 111 (76%) 34 (24%) 39.2 ± 14.8 40.5 ± 14.1 36.1 ± 16.2
342 146 (43%) 196 (57%) 279 (82%) 63 (18%) 41.3 ± 13.1 39.2 ± 13.4 44.1 ± 12.1
RHD patients
RFo patients
103 25 (24.3%) 78 (75.8%) 86 (83.5%) 17 (16.5%) 46.4 ± 10.8 46.1 ± 10.7 47.2 ± 11.1
42 18 (42.8%) 24 (57.1%) 25 (59.5%) 17 (40.5%) 21.6 ± 6.0 22.3 ± 5.8 20.7 ± 6.3
OR [95% CI]a
P valuea
OR [95% CI]b
P valueb
0.57 [0.37–0.86]
G
n.a. p.R99Q p.D120G
n.a. CUB1 CUB1
Variable P600 ng/ml 6200 ng/ml
100 (34.5) 0 5 (1.7)
215 (31.4) 6 (0.9) 8 (1.2)
76 (36.9) 0 4 (1.9)
24 (28.6) 0 1 (1.2)
1 2 2 3 3 3 4
rs56392418 rs2273344 rs9430347 rs17409276 rs12711521 rs2273346 rs12085877
Exon 3 Intron 4 Intron 5 Intron 9 Exon 10 Exon 10 Exon 12
g.5638C>T g.7164A>G g.7441G>A g.21081C>T g.21370G>T g.21389T>C g.24599G>A
p.P126L n.a. n.a. n.a. p.Y371D p.V377A p.R439H
CUB1 n.a. n.a. n.a. CCP2 CCP2 SP
6200 ng/ml P600 ng/ml P600 ng/ml P600 ng/ml Variable 6200 ng/ml 6200 ng/ml
6 (2.1) 62 (21.4) 62 (21.4) 51 (17.6) 81 (27.9) 6 (2.1) 0
9 (1.3) 124 (18.1) 124 (18.1) 103 (15.1) 183 (26.8) 32 (4.7) 6 (0.9)
6 (2.9) 44 (21.4) 44 (21.4) 36 (17.5) 61 (29.6) 3 (1.5) 0
0 18 (21.4) 18 (21.4) 15 (17.9) 20 (23.8) 3 (3.6) 0
4
rs1782455
Exon 12
g.24762T>C
p.S493=
SP
Variable
n.a. Normal Cannot bind MBL, cannot activate C4 Normal n.a. n.a. n.a. Normal Normal Binds MBL but does not autoactivate, cannot activate C4 n.a. (synonymous)
74 (25.5)
149 (21.8)
57 (27.7)
17 (20.2)
Haplotype block
dbSNP
1 1 1
Gene region
Reference: NG007289.1 alleles
dbSNP: database of single nucleotide polymorphisms; n: number of chromosomes; RHD: rheumatic heart disease; RFo: rheumatic fever only; n.a.: not applicable; CUB1: C1r/ C1s, UegF and bone morphogenetic protein 1; CCP2: complement control protein; SP: serine protease. In bold: alleles negatively associated with the disease. Allele nomenclature was italicized, as recommended by the HGVS. a Reported effect of homozygosity of exonic minor alleles [16–19] and intronic minor alleles [18,19]. b Reported results from in vitro studies [17]. Allele frequencies are given as percentages in parentheses. Haplotype blocks: (1) promoter variant at nucleotide position 1945560 and three amino acid variants due to nucleotide substitutions at codons 99, 120 and 126 (ARDP, ARGP, CRDP, CQDP, CRDL); (2) intron 4 and 5 variants (AG, GA, GG), (3) intron 9 and two amino acid variants at codons 371 and 377 (CDA, CDV, TDV), (4) amino acid variant at codon 439 and a synonymous variant at nucleotide position 24762 (RC, HC, RT).
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Table 3 MASP2 haplotype frequencies (%) in patients and controls. Haplotypea ARDP AG CYV RT ARDP GA CYV RT ARGP AG CYV RT CQDP GA TDV RC CRDP AG CYV RT CRDP AG TDV RC CRDP GA CYV RT CRDP GA TDV RC ARDP AG TDV RC Protective haplotypes CRDL AG CDV HC CRDP AG CDA RT Susceptibility haplotypes CRDL AG CDV RC CRDP AG CDV RC CRDP AG CDV RT CRDP GA CDV RC
Phylogenetic nomenclatureb
Protein Levelsc
⁄
2B2A-i [AG] 2B2A-i [GA] 2B2B-l [AG] ⁄ 1B2-h [GA] ⁄ 2B1-i [AG] ⁄ 1B1-h [AG] ⁄ 2B1-i [GA] ⁄ 1B1-h [GA] ⁄ 2B2A-i.1B1-h [AG]
>200 < 400 ng/ml >400 < 600 ng/ml 6200 ng/ml P600 ng/ml >200 < 400 ng/ml P400 ng/ml >400 < 600 ng/ml P600 ng/ml >400 < 600 ng/ml
Normal Normal Deficient Normal Normal Normal Normal Normal Normal
⁄
1C2-l [AG] 2A2-l [AG]
6200 ng/ml 6200 ng/ml
Deficient Normal
1C1-l [AG] 1A [AG] ⁄ 2A1 [AG] ⁄ 1A [GA]
6200 ng/ml Variable Variable Variable
Normal Normal Normal Normal
⁄ ⁄
⁄
⁄ ⁄
C4 activation
d
d
Patients (n = 290)
Controls (n = 684)
RHD patients (n = 206)
RFo patients (n = 84)
181 (62.4) 3 (1.0) 5 (1.7) 0 13 (4.5) 2 (0.7) 7 (2.4) 48 (16.6) 1 (0.3)
453 (66.2) 7 (1.0) 8 (1.2) 6 (0.9) 22 (3.2) 5 (0.7) 11 (1.6) 91 (13.3) 1 (0.1)
124 (60.2) 2 (1.0) 4 (1.9) 0 11 (5.3) 2 (1.0) 4 (1.9) 34 (16.5) 0
57 (67.9) 1 (1.2) 1 (1.2) 0 2 (2.4) 0 3 (3.6) 14 (16.7) 1 (1.2)
0 6 (2.1)
6 (0.9) 28 (4.7)
0 3 (1.5)
0 3 (3.6)
6 (2.1) 13 (4.5) 1 (0.3) 4 (1.4)
3 (0.4) 28 (4.1) 2 (0.3) 9 (1.3)
6 (2.9) 11 (5.3) 1 (0.5) 4 (1.9)
0 2 (2.4) 0 0
Haplotype nomenclature was italicized, as recommended by the HGVS. Haplotype frequencies are given as percentages in parentheses. n: number of chromosomes, RHD: rheumatic heart disease; RFo: rheumatic fever only. a Each haplotype block is separated by a space. First haplotype block include variants from the promoter to exon 3, second block include polymorphisms in intron 4 and 5, third block include variants in intron 9 and exon 10, fourth block include variants in exon 12. For nonsynonimous SNPs, aminoacid changes are indicated, e.g. CDV means a cytosine (C) at position 21081 in intron 9, followed by two closely located nucleotide substitutions encoding asparagine (D) at residue 371 and valine (V) at residue 377 of the protein. b In the alphanumerical system of the phylogenetic nomenclature, first clade is given by a number, followed by as many letters and numbers as branches/lineages in the tree [39]. In the case of MASP2, the system was added by a ‘‘l’’ for haplotypes generating ‘‘low’’ MASP-2 levels, ‘‘i’’ for haplotypes generating ‘‘intermediate’’ MASP-2 levels, ‘‘h’’ for haplotypes generating ‘‘high’’ MASP-2 levels [18]. It was also added by the alleles in introns 4 and 5 (within brackets), which are not in linkage disequilibrium with all other investigated SNPs [19]. Recombinant haplotypes are given by the names of the possible parental haplotypes, separated by a dot. Clade 1 haplotypes share g.24762C in exon 12: ⁄1A (most ancient), ⁄1B1-h and ⁄1B2-h (sharing g.21081T), ⁄1C1-l and ⁄1C2-l (sharing p.126L). Five other haplotypes present g.24762T in exon 12 and belong to clade 2: ⁄ 2A1, ⁄2A2-l (with the p.377A variant), ⁄2B1-I, the very common ⁄2B2A-i and ⁄2B2B-l (with the p.120G variant). c Reported effect of homozygous haplotypes [18,19]. d Reported results from in vitro studies. The effect of a recombinant protein with both p.126L and p.439H residues, encoded by ⁄1C2-l, is unknown, although a protein with p.439H is unable to bind MBL [17].
[AG]) increased the susceptibility to RHD among RF patients (22/206 or 10.7% in RHD patients vs. 2/84 or 2.4% in RFo patients, P = 0.02, OR = 4.9 [95% CI = 1.13–21.34]). This association remained after correction for age, which was the only demographic factor associated with RHD in comparison to RFo patients (P = 0.02).
and ethnic group: P < 0.0001, OR = 0.20 [95% CI = 0.09–0.47]) (Fig. 1). The difference remained after comparing controls and RHD, but not RFo patients, and was independent of sex, age and
3.2. MASP-2 levels Patients presented lower MASP-2 levels than controls, with medians 252.8 and 313.9 ng/ml, respectively (corrected for age
Fig. 1. MASP-2 levels in patients and controls. Medians and min–max ranges are shown. MASP-2 levels were compared with Mann–Whitney test.
Fig. 2. MASP-2 levels are associated with MASP2 genotypes. Medians and min–max ranges are shown. MASP-2 levels were compared with Kruskal–Wallis test. h: genotypes with haplotypes containing the suffix h (h/h and h/i), ii: homozygote genotypes with haplotypes containing the suffix i (i/i), l: genotypes with haplotypes containing the suffix l (l/l and l/i). We excluded 41 individuals (22 controls and 19 patients) due to ambiguous genotype–phenotype associations: seven were h/l heterozygotes and the others presented haplotypes not formerly associated with MASP-2 levels [18,19].
S.J.d.S. Catarino et al. / Human Immunology 75 (2014) 1197–1202
ancestry (P < 0.0001, OR = 0.11 [95% CI = 0.03–0.42]). MASP-2 levels, nevertheless, did not differ between RHD and RFo patients (medians 254.9 and 241.6 ng/ml, respectively). We further confirmed previously reported associations of MASP2 haplotypes with MASP-2 levels, in both controls and patients. This was evident comparing genotypes with haplotypes reportedly associated with high MASP-2 concentrations (equal or higher than 600 ng/ml, produced by haplotypes containing the suffix h: h/h and h/i), intermediate concentrations (between 200 and 600 ng/ml, produced by haplotype with the suffix i: i/i) and low concentrations (less than or equal to 200 ng/ml, produced by haplotype with the suffix l: l/l and l/i). As expected, the genotype–phenotype association was more conspicuous in the control group (Fig. 2). Interestingly, we did not identify p.99Q among patients, which is a variant known to be associated with high MASP-2 levels (0.9% in controls, not significant) (Table 2).
4. Discussion Rheumatic fever is still a disease with great impact on the public health system of developing countries, where disease prevalence is high and expenses with cardiac surgeries, expressive [28]. Thus, attempts to elucidate the autoimmune and physiological mechanisms in this condition are important. Protection against invading pathogens relies on complex interactions between the genetically controlled innate and adaptive immune responses. In fact, several polymorphisms in genes that encode molecules involved in both innate and adaptive immune responses were shown to contribute to RF and RHD susceptibility [29]. The activation of complement cascade provides a first line of defense against Streptococcus pyogenes infections. Due to its importance in clearance of rheumatic etiological agents as well as in disposal of apoptotic bodies and potential autoimmune initiators, deficiencies of components of the lectin pathway have been found to increase susceptibility and modulate severity of most rheumatic disorders [30]. This is the first study to investigate a number of polymorphisms encompassing the whole MASP2 gene as well as related haplotypes and MASP-2 levels in patients with RF and RHD. We confirmed previously noticed associations between MASP2 polymorphisms/ haplotypes and MASP-2 levels [16–19] and the absence of an association with the p.D120G polymorphism [26,31]. MASP-2 levels were lower in patients, than in controls. These patients were the same formerly found with higher MBL levels [32]. Higher MBL and lower MASP-2 levels are consistent with MASP-2 consumption due to intense MBL-driven complement activation. Lower MASP-2 levels were also found in patients with myocardial infarction compared to controls, suggesting an involvement of the protein in complement activation following ischemia and myocardial necrosis [33]. On the other hand, higher MASP-2 levels were associated with improved survival in patients with hematologic malignancies, specifically lymphoma [34]. We further found an association of two SNPs (p.V377A and p.R439H) known to cause low MASP-2 levels [16,17], with protection against RF and RHD. p.R439H was reported also to protect against placental malaria [21]. In contrast, these SNPs were recently described to increase the susceptibility to leprosy [19]. The contrasting associations are not surprising, since MASP-2 modulates phagocytosis, complement and coagulation cascades, each exerting a different role in the susceptibility to infectious and autoimmune diseases [8,16,17]. Furthermore, the association between variants leading to low protein levels and protection against RF has been formerly found for MBL2 [35,36], but not for FCN2 [37]. The low basal concentrations of MBL and MASP-2 correspond to a lower capacity of complement activation, due to structural variants as p.52C, p.54D and p.57E in MBL2 and p.439H in MASP2 [30].
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This reinforces our suggestion that complement activation through the lectin pathway promote the inflammatory response and subsequent tissue damage in rheumatic fever disease. On the other hand, MASP2 CDVR haplotypes, encompassing g.1961795C, p.371D, p.377V and p.439R variants, where associated with an almost five times increased risk to RHD. They generate varying MASP-2 levels and are phylogenetically related to the most ancestral MASP2 haplotype [18]. These haplotypes probably harbor other polymorphisms not investigated in this study, which could contribute to the disease. Interestingly, the p.371D variant was also reported to increase susceptibility to HCV infection [20], D/D homozygotes were associated with bacterial infections after orthotopic liver transplantation [22] and the CD haplotype was found associated with susceptibility to chagasic cardiomyopathy [23]. Although cardiac commitments in Chagas disease and RHD cannot be compared, both may share autoimmune etiology [38]. In conclusion, the effects of MASP2 polymorphisms on protein serum levels and functional efficiency may modulate susceptibility to RF. MASP-2 levels were lower in patients, which may reflect protein consumption due to complement activation. This is in agreement with our former results regarding MBL protein and polymorphisms of the MBL2 gene. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD. Funding This work was supported by CNPq - Brazil (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES - Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Acknowledgments We gratefully acknowledge the patients for their participation in this study and thank the staff of the Laboratório de Imunopatologia Molecular HC/UFPR for their assistance. References [1] Chang C. Cutting edge issues in rheumatic fever. Clin Rev Allergy Immunol 2011:1–25. [2] Burke RJ, Chang C. Diagnostic criteria of acute rheumatic fever. Autoimmun Rev 2014;13:503–7. [3] Guilherme L, Kohler K, Kalil J. Rheumatic heart disease: mediation by complex immune events. Adv Clin Chem 2011;53:31–50. [4] Tibazarwa K, JA V, Mayosi B. Incidence of acute rheumatic fever in the world: a systematic review of population-based studies. Heart 2008;94:1534–40. [5] Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685–94. [6] Kumar RK, Tandon R. Rheumatic fever & rheumatic heart disease: the last 50 years. Indian J Med Res 2013;137:643–58. [7] Barbosa P, Muller R, Latado A, Achutti A, Ramos A, Weksler C. Diretrizes brasileiras para o diagnóstico, tratamento e prevenção da febre reumática. Arq Bras Cardiol 2009;93:1–18. [8] Mayilyan KR. Complement genetics, deficiencies, and disease associations. Protein Cell 2012;3:487–96. [9] Stäger S, Alexander J, Kirby AC, Botto M, Van Rooijen N, Smith DF, et al. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat Med 2003;9:1287–92. [10] Degn SE, Jensenius JC, Thiel S. Disease-causing mutations in genes of the complement system. Am J Hum Genet 2011;88:689–705. [11] Matsushita M, Endo Y, Fujita T. Structural and functional overview of the lectin complement pathway: its molecular basis and physiological implication. Arch Immunol Ther Exp (Warsz) 2013;61:273–83. [12] Stover CM, Thiel S, Thelen M, Lynch NJ, Vorup-Jensen T, Jensenius JC, et al. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene. J Immunol 1999;162:3481–90. [13] Thiel S. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins. Mol Immunol 2007;44:3875–88. [14] Laursen I, Thielens N, Christiansen M, Houen G. MASP interactions with plasma-derived MBL. Mol Immunol 2012;52:79–87.
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[15] Stengaard-Pedersen K, Thiel S, Gadjeva M, Møller-Kristensen M, Sørensen R, Jensen LT, et al. Inherited deficiency of mannan-binding lectin-associated serine protease 2. N Engl J Med 2003;349:554–60. [16] Thiel S, Steffensen R, Christensen IJ, Ip WK, Lau YL, Reason IJM, et al. Deficiency of mannan-binding lectin associated serine protease-2 due to missense polymorphisms. Genes Immun 2007;8:154–63. [17] Thiel S, Kolev M, Degn S, Steffensen R, Hansen AG, Ruseva M, et al. Polymorphisms in mannan-binding lectin (MBL)-associated serine protease 2 affect stability, binding to MBL, and enzymatic activity. J Immunol 2009;182:2939–47. [18] Boldt ABW, Grisbach C, Steffensen R, Thiel S, Kun JFJ, Jensenius JC, et al. Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19 serum levels. Hum Immunol 2011;72:753–60. [19] Boldt ABW, Goeldner I, Stahlke ERS, Thiel S, Jensenius JC, de Messias-Reason IJT. Leprosy association with low MASP-2 levels generated by MASP2 haplotypes and polymorphisms flanking MAp19 exon 5. PLoS One 2013;8:e69054. [20] Tulio S, Faucz FR, Werneck RI, Olandoski M, Alexandre RB, Boldt ABW, et al. MASP2 gene polymorphism is associated with susceptibility to hepatitis C virus infection. Hum Immunol 2011;72:912–5. [21] Holmberg V, Onkamo P, Lahtela E, Lahermo P, Bedu-Addo G, Mockenhaupt FP, et al. Mutations of complement lectin pathway genes MBL2 and MASP2 associated with placental malaria. Malar J 2012;11:61. [22] De Rooij B-JF, van Hoek B, ten Hove WR, Roos A, Bouwman LH, Schaapherder AF, et al. Lectin complement pathway gene profile of donor and recipient determine the risk of bacterial infections after orthotopic liver transplantation. Hepatology 2010;52:1100–10. [23] Boldt ABW, Luz PR, Messias-Reason IJT. MASP2 haplotypes are associated with high risk of cardiomyopathy in chronic Chagas disease. Clin Immunol 2011;140:63–70. [24] Goeldner I, Skare T, Boldt ABW, Nass FR, Messias-Reason IJ, Utiyama SR. Association of MASP-2 levels and MASP2 gene polymorphisms with rheumatoid arthritis in patients and their relatives. PLoS One 2014;9:e90979. [25] Dajani AS, Ayoub E, Bierman FZ, Bisno AL, Denny FW, Durack DT, et al. Guidelines for the diagnosis of rheumatic fever: Jones criteria, 1992 update. JAMA 1992;268:2069–73. [26] Schafranski MD, Pereira Ferrari L, Scherner D, Torres R, de Messias-Reason IJ. Functional MASP2 gene polymorphism in patients with history of rheumatic fever. Hum Immunol 2008;69:41–4.
[27] Excoffier L, Lischer HEL. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 2010;10:564–7. [28] Guilherme L, Kalil J. Rheumatic heart disease: molecules involved in valve tissue inflammation leading to the autoimmune process and anti-S. pyogenes vaccine. Front Immunol 2013;4:352. [29] Guilherme L, Köhler KF, Kalil J. Rheumatic heart disease: genes, inflammation and autoimmunity. Rheumatol Curr Res 2012;S4(001):1–5. [30] Boldt ABW, Goeldner I, De Messias-Reason IJT. Relevance of the lectin pathway of complement in rheumatic diseases. Adv Clin Chem 2012;56:105–53. [31] Ramasawmy R, Spina GS, Fae KC, Pereira AC, Nisihara R, Messias Reason IJ, et al. Association of mannose-binding lectin gene polymorphism but not of mannose-binding serine protease 2 with chronic severe aortic regurgitation of rheumatic etiology. Clin Vaccine Immunol 2008;15:932–6. [32] Schafranski MD, Pereira Ferrari L, Scherner D, Torres R, Jensenius JC, de Messias-Reason IJ. High-producing MBL2 genotypes increase the risk of acute and chronic carditis in patients with history of rheumatic fever. Mol Immunol 2008;45:3827–31. [33] Zhang M, Hou YJ, Cavusoglu E, Lee DC, Steffensen R, Yang L, et al. MASP-2 activation is involved in ischemia-related necrotic myocardial injury in humans. Int J Cardiol 2013;166:499–504. [34] Zehnder A, Fisch U, Hirt A, Niggli FK, Simon A, Ozsahin H, et al. Prognosis in pediatric hematologic malignancies is associated with serum concentration of mannose-binding lectin-associated serine protease-2 (MASP-2). Pediatr Blood Cancer 2009;53:53–7. [35] Schafranski MD, Stier A, Nisihara R, Messias-Reason IJT. Significantly increased levels of mannose-binding lectin (MBL) in rheumatic heart disease: a beneficial role for MBL deficiency. Clin Exp Immunol 2004;138:521–5. [36] Messias Reason IJ, Schafranski MD, Jensenius JC, Steffensen R. The association between mannose-binding lectin gene polymorphism and rheumatic heart disease. Hum Immunol 2006;67:991–8. [37] Messias-Reason IJ, Schafranski MD, Kremsner PG, Kun JFJ. Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease. Clin Exp Immunol 2009;157:395–9. [38] McLean B, Oudit GY. Role of autoimmunity in heart disease: is Chagas heart disease the definitive proof? Can J Cardiol 2014;30:267–9. [39] Nebert DW. Proposal for an allele nomenclature system based on the evolutionary divergence of haplotypes. Hum Mutat 2002;20:463–72.
Contents lists available at ScienceDirect
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Association of MASP2 polymorphisms and protein levels with rheumatic fever and rheumatic heart disease Sandra Jeremias dos Santos Catarino a, Angelica Beate Winter Boldt a,b, Marcia Holsbach Beltrame a, Renato Mitsunori Nisihara a, Marcelo Derbli Schafranski c, Iara Jose de Messias-Reason a,⇑ a b c
Laboratório de Imunopatologia Molecular, Departamento de Patologia Médica, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, Brazil Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brazil Departamento de Medicina, Universidade Estadual de Ponta Grossa, Paraná, Brazil
a r t i c l e
i n f o
Article history: Received 21 March 2014 Accepted 6 October 2014 Available online 12 October 2014 Keywords: Rheumatic fever Rheumatic heart disease MASP2 Polymorphism Haplotype-specific genotyping
a b s t r a c t MASP-2 is a key protein of the lectin pathway of complement system. Several MASP2 polymorphisms were associated with MASP-2 serum levels or functional activity. Here we investigated a possible association between MASP2 polymorphisms and MASP-2 serum levels with the susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD). We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 145 patients with history of RF from south Brazil (103 with RHD and 42 without cardiac lesion [RFo]) and 342 healthy controls. MASP-2 levels were determined by ELISA. The low MASP-2 producing p.377A and p.439H variants were negatively associated with RF (P = 0.02, OR = 0.36) and RHD (P = 0.01, OR = 0.25). In contrast, haplotypes that share the intron 9 – exon 12 g.1961795C, p.371D, p.377V and p.439R polymorphisms increased the susceptibility to RHD (P = 0.02, OR = 4.9). MASP-2 levels were associated with MASP2 haplotypes and were lower in patients (P < 0.0001), which may reflect protein consumption due to complement activation. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatic fever (RF) and its most severe sequel chronic rheumatic heart disease (RHD) are chronic inflammations that follow oropharynx infection by b-hemolytic Streptococcus group A. The disease occurs in genetically predisposed children and teenagers (aged 3–19 years) affecting the heart, joints, nervous system and skin [1]. The onset of RF usually occurs two to three weeks following the initial pharyngitis, but in some cases the onset may be months later [2]. Carditis is the most severe clinical manifestation of RF, affecting about 30–50% of patients 4–8 weeks after the first RF episode [3]. In general, carditis progress to RHD, associated with chronic inflammation and stenosis of valve tissue, leading to permanent heart damage. RHD affects young adults and remains a
Abbreviations: MASP-2, mannan-binding lectin-associated serine protease 2; MASP2, MASP-2 gene; MBL, mannan-binding lectin; RF, rheumatic fever; RHD, rheumatic heart disease. ⇑ Corresponding author at: Laboratório de Imunopatologia Molecular, Serviço de Anatomia Patológica, Hospital de Clínicas, Federal University of Paraná (UFPR), R. General Carneiro, 181, CEP 80060-900 Curitiba, PR, Brazil. E-mail address: [email protected] (I.J. de Messias-Reason).
major public health problem in Brazil and other developing countries, due to high morbidity and mortality. RF incidence exceeds 50 per 100,000 children in some developing countries [4] and RHD global prevalence varies between 15 and 20 million cases [5]. About two million cases require repeated hospitalization and one million may need a heart transplant in 5–20 years, generating high costs to the health system [1,5,6]. In Brazil, the estimated rate is 10 million cases of streptococcal pharyngitis each year, resulting in 30,000 new cases of RF, of which approximately 15,000 progress to RHD [7]. The complement system serves as the backbone of innate immunity and supports the adaptive immune system in gaining momentum to respond. At present, more than 40 components of complement have been described [8]. The complement system is activated through the classical, alternative and lectin pathways. Complement activation leads to recruitment of inflammatory mediators, pathogen destruction and clearance of immune complexes and apoptotic cells [9,10]. The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrates or acetylated residues on the surface of pathogens, respectively [11]. MBL and ficolins are associated with serine
http://dx.doi.org/10.1016/j.humimm.2014.10.003 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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proteases named MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which mediate the formation of C3 convertase [11]. MASP-2 and MAp19 (also known as sMAP or small MAP) are encoded by the MASP2 gene, located on 1p36.23-31. MAp19 is a truncated protein resulting from alternative splicing and inclusion of exon 5 in the mature mRNA [12]. MASP-2 and MAp19 share two domains encoded by the first four exons (CUB1 – C1r/C1s, Uegf and bone morphogenetic protein-1 – and an epidermal growth factor (EGF)-like domain). Exon 5 encodes the four last amino acids exclusive of MAp19. Exons 6–11 encode a second CUB domain (CUB2) and two contiguous complement control protein modules (CCP1 and CCP2). Exon 12 encodes the activating peptide and serine protease domain [13]. MASP-2 plays a key role in the activation of the lectin pathway initiated by ficolins, colectin 11 or MBL. MAp19 binds MBL, but its function remains speculative [14]. MASP-2 deficiency was first described in a patient with multiple infections and autoimmune manifestations, due to an exon 3 mutation causing the exchange of aspartic acid with glycine at position 120 (p.D120G) [15]. Several other MASP2 polymorphisms, including g.1945560C>A in the promoter region, p.R99Q and p.P126L in exon 3, g.7164A>G in intron 4, g.7441G>A in intron 5, g.1961795C>T in intron 9, p.D371Y and p.V377A in exon 10, p.R439H and g.24762C>T in exon 12, were found associated with serum levels or functional activity of MASP-2 [16–19]. Some of them (p.126L, p.377A, p.439H – associated with low MASP-2 levels and p.371D – associated with high MASP-2 levels probably due to linkage disequilibrium with intronic variants) were associated with susceptibility to diseases: leprosy [19], hepatitis C [20], malaria [21], bacterial infections after orthotopic liver transplantation [22], Chagas disease [23] and rheumatoid arthritis [24]. These SNPs are distributed in ten main haplotypes comprised of four variant blocks. The first block contains the promoter variant at nucleotide position 1945560 and three amino acid variants at codons 99, 120 and 126 (ARDP, ARGP, CRDP, CQDP, CRDL); the second block contains the intron 4 and 5 variants (AG, GA, GG), the third block by the intron 9 and two amino acid variants at codons 371 and 377 (CDA, CDV, TDV), fourth block contains one amino acid variant at codon 439 and a synonymous variant at nucleotide position 24762 (RC, HC, RT). For practical reasons, the haplotypes were named according to their phylogenetic relationships. Five haplotypes belong to clade 1 and share g.24762C in exon 12. Among them, ⁄1A represents the most ancient haplotype, ⁄1B1-h and ⁄ 1B2-h share g.1961795T in intron 9 and are associated with higher MASP-2 levels (reason for the ‘‘h’’ suffix), ⁄1C1-l and ⁄1C2-l share p.126L and are associated with low MASP-2 levels (reason for the ‘‘l’’ suffix). Importantly, ⁄1C2-l also bears the deficiency-causing p.439H variant. Five other haplotypes belong to clade 2 and present g.24762T in exon 12: ⁄2A1, ⁄2A2-l (with p.377A), ⁄2B1-i and ⁄ 2B2A-i (both associated with intermediate – ‘‘i’’ – MASP-2 levels), and the MASP-2 deficiency-causing ⁄2B2B-l haplotype (with the p.120G variant) [18]. Variants in introns 4 and 5 mainly occur as two combinations in cis, AG and GA, in the ⁄1A, ⁄1B1-h, ⁄2A1, ⁄ 2B1-I and ⁄2B2A-I haplotypes, reason for which they are added between clasps after the haplotype name (e.g. ⁄1A [AG]). These polymorphisms probably modulate alternative exon 5 splicing, since GA is associated with higher MASP-2 and lower MAp19 levels, the opposite being true for AG [19].
2. Material and methods 2.1. Subjects and samples This study was approved by the local ethics committee (CEP/HC 2658.265/2011-11). We investigated a total of 145 patients with a
history of RF with mean age of 39 years (range = 18–89 years). All patients had a history of RF and were diagnosed according to Jones’ modified criteria [25]. Among them, 103 (71%) had RHD, confirmed by transthoracic echocardiogram showing rheumatic involvement of the mitral valve; 42 patients (29%) did not present RHD but had history of RF and were designated as ‘‘rheumatic fever only’’ (RFo) patients. Clinical characterization of RFo and RHD patients was described in a previous study [26]. None of the patients presented other inflammatory disease, neoplasia, infective endocarditis or other infection at the time of blood collection. The control group included 286 blood donors and 56 health workers and other volunteers (without history of rheumatic fever) from the same geographic region, with a mean age of 41.3 years (range = 18–61 years). Among the blood donors, 65 were from Hospital de Clínicas of the UFPR, 174 were from Centro de Hemoterapia e Hematologia do Paraná (Hemepar) and 47 from Biobanco of the Hospital Evangélico, paired with the patients according to age and ancestry. Differences in sex distribution between patients and controls and in age distribution between RHD and RFo patients were corrected with multivariate logistic regression (Table 1). 2.2. MASP2 genotyping A total of eleven MASP2 single nucleotide polymorphisms (SNPs) were investigated. Taking NG007289.1 as reference sequence, they were: g.4847A>C in the promoter (rs7548659), g.5557G>A (rs61735600), g.5620A>G (rs72550870) and g.5638C>T (rs56392418) in exon 3 (causing amino acid substitutions p.R99Q, p.D120G and p.P126L in the CUB1 domain, respectively), g.7164A>G (rs2273344) in intron 4, g.7441G>A (rs9430347) in intron 5, g.21081C>T in intron 9 (rs17409276), g.21370G>T (rs12711521) and g.21389T>C (rs2273346) in exon 10 (causing amino acid substitutions p.Y371D and p.V377A in the CCP2 domain, respectively) and g.24599G>A (rs12085877) and g.24762T>C (rs1782455) in exon 12 (encoding one non synonymous – p.R439H and one synonymous variant – p.S493=). Minor intronic alleles (g.7164G, g.7441A and g.21081T) have been formerly associated with MASP-2 levels higher than 600 ng/ml [18,19], whereas minor non synonymous alleles encoding p.120G, p.126L, p.377A and p.439H were associated with MASP-2 levels lower than 200 ng/ml. Among them, homozygotes for p.120G or p.439H are unable to activate the lectin pathway of complement [16,17]. The SNPs were identified by a multiplex sequence-specific amplification method (multiplex PCR-SSP), as previously described [18,19]. 2.3. MASP-2 assay MASP-2 concentrations were measured in the sera of 145 patients (42 RFo and 103 RHD) and 196 controls using enzymelinked immunosorbent assay (HK326, Hycult Biotechnology, Uden, The Netherlands). Both groups were homogeneous regarding MASP2 genotype distribution. Minimum concentration which can be measured is 1.6 ng/ml. Color intensity was evaluated at 450 nm in an ELISA reader. 2.4. Statistics Genotype, allele and haplotype frequencies were obtained by direct counting. SNPs distributed from the promoter to exon 12 were phased with the SSP primers. In most cases, the phase between distantly situated SNPs could be deduced due to strong linkage disequilibrium between the variants [18]. Hardy– Weinberg equilibrium and homogeneity between genotype distributions were tested using ARLEQUIN software package version 3.5.1.3 [27]. MASP-2 levels were tested for normality
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n Male (%) Female (%) Euro-Brazilians (%) Afro-Brazilians (%) Mean age ± SD Mean age female ± SD Mean age male ± SD
n Male (%) Female (%) Euro-Brazilians (%) Afro-Brazilians (%) Mean age ± SD Mean age female ± SD Mean age male ± SD
Patients
Controls
145 43 (30%) 102 (70%) 111 (76%) 34 (24%) 39.2 ± 14.8 40.5 ± 14.1 36.1 ± 16.2
342 146 (43%) 196 (57%) 279 (82%) 63 (18%) 41.3 ± 13.1 39.2 ± 13.4 44.1 ± 12.1
RHD patients
RFo patients
103 25 (24.3%) 78 (75.8%) 86 (83.5%) 17 (16.5%) 46.4 ± 10.8 46.1 ± 10.7 47.2 ± 11.1
42 18 (42.8%) 24 (57.1%) 25 (59.5%) 17 (40.5%) 21.6 ± 6.0 22.3 ± 5.8 20.7 ± 6.3
OR [95% CI]a
P valuea
OR [95% CI]b
P valueb
0.57 [0.37–0.86]
G
n.a. p.R99Q p.D120G
n.a. CUB1 CUB1
Variable P600 ng/ml 6200 ng/ml
100 (34.5) 0 5 (1.7)
215 (31.4) 6 (0.9) 8 (1.2)
76 (36.9) 0 4 (1.9)
24 (28.6) 0 1 (1.2)
1 2 2 3 3 3 4
rs56392418 rs2273344 rs9430347 rs17409276 rs12711521 rs2273346 rs12085877
Exon 3 Intron 4 Intron 5 Intron 9 Exon 10 Exon 10 Exon 12
g.5638C>T g.7164A>G g.7441G>A g.21081C>T g.21370G>T g.21389T>C g.24599G>A
p.P126L n.a. n.a. n.a. p.Y371D p.V377A p.R439H
CUB1 n.a. n.a. n.a. CCP2 CCP2 SP
6200 ng/ml P600 ng/ml P600 ng/ml P600 ng/ml Variable 6200 ng/ml 6200 ng/ml
6 (2.1) 62 (21.4) 62 (21.4) 51 (17.6) 81 (27.9) 6 (2.1) 0
9 (1.3) 124 (18.1) 124 (18.1) 103 (15.1) 183 (26.8) 32 (4.7) 6 (0.9)
6 (2.9) 44 (21.4) 44 (21.4) 36 (17.5) 61 (29.6) 3 (1.5) 0
0 18 (21.4) 18 (21.4) 15 (17.9) 20 (23.8) 3 (3.6) 0
4
rs1782455
Exon 12
g.24762T>C
p.S493=
SP
Variable
n.a. Normal Cannot bind MBL, cannot activate C4 Normal n.a. n.a. n.a. Normal Normal Binds MBL but does not autoactivate, cannot activate C4 n.a. (synonymous)
74 (25.5)
149 (21.8)
57 (27.7)
17 (20.2)
Haplotype block
dbSNP
1 1 1
Gene region
Reference: NG007289.1 alleles
dbSNP: database of single nucleotide polymorphisms; n: number of chromosomes; RHD: rheumatic heart disease; RFo: rheumatic fever only; n.a.: not applicable; CUB1: C1r/ C1s, UegF and bone morphogenetic protein 1; CCP2: complement control protein; SP: serine protease. In bold: alleles negatively associated with the disease. Allele nomenclature was italicized, as recommended by the HGVS. a Reported effect of homozygosity of exonic minor alleles [16–19] and intronic minor alleles [18,19]. b Reported results from in vitro studies [17]. Allele frequencies are given as percentages in parentheses. Haplotype blocks: (1) promoter variant at nucleotide position 1945560 and three amino acid variants due to nucleotide substitutions at codons 99, 120 and 126 (ARDP, ARGP, CRDP, CQDP, CRDL); (2) intron 4 and 5 variants (AG, GA, GG), (3) intron 9 and two amino acid variants at codons 371 and 377 (CDA, CDV, TDV), (4) amino acid variant at codon 439 and a synonymous variant at nucleotide position 24762 (RC, HC, RT).
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Table 3 MASP2 haplotype frequencies (%) in patients and controls. Haplotypea ARDP AG CYV RT ARDP GA CYV RT ARGP AG CYV RT CQDP GA TDV RC CRDP AG CYV RT CRDP AG TDV RC CRDP GA CYV RT CRDP GA TDV RC ARDP AG TDV RC Protective haplotypes CRDL AG CDV HC CRDP AG CDA RT Susceptibility haplotypes CRDL AG CDV RC CRDP AG CDV RC CRDP AG CDV RT CRDP GA CDV RC
Phylogenetic nomenclatureb
Protein Levelsc
⁄
2B2A-i [AG] 2B2A-i [GA] 2B2B-l [AG] ⁄ 1B2-h [GA] ⁄ 2B1-i [AG] ⁄ 1B1-h [AG] ⁄ 2B1-i [GA] ⁄ 1B1-h [GA] ⁄ 2B2A-i.1B1-h [AG]
>200 < 400 ng/ml >400 < 600 ng/ml 6200 ng/ml P600 ng/ml >200 < 400 ng/ml P400 ng/ml >400 < 600 ng/ml P600 ng/ml >400 < 600 ng/ml
Normal Normal Deficient Normal Normal Normal Normal Normal Normal
⁄
1C2-l [AG] 2A2-l [AG]
6200 ng/ml 6200 ng/ml
Deficient Normal
1C1-l [AG] 1A [AG] ⁄ 2A1 [AG] ⁄ 1A [GA]
6200 ng/ml Variable Variable Variable
Normal Normal Normal Normal
⁄ ⁄
⁄
⁄ ⁄
C4 activation
d
d
Patients (n = 290)
Controls (n = 684)
RHD patients (n = 206)
RFo patients (n = 84)
181 (62.4) 3 (1.0) 5 (1.7) 0 13 (4.5) 2 (0.7) 7 (2.4) 48 (16.6) 1 (0.3)
453 (66.2) 7 (1.0) 8 (1.2) 6 (0.9) 22 (3.2) 5 (0.7) 11 (1.6) 91 (13.3) 1 (0.1)
124 (60.2) 2 (1.0) 4 (1.9) 0 11 (5.3) 2 (1.0) 4 (1.9) 34 (16.5) 0
57 (67.9) 1 (1.2) 1 (1.2) 0 2 (2.4) 0 3 (3.6) 14 (16.7) 1 (1.2)
0 6 (2.1)
6 (0.9) 28 (4.7)
0 3 (1.5)
0 3 (3.6)
6 (2.1) 13 (4.5) 1 (0.3) 4 (1.4)
3 (0.4) 28 (4.1) 2 (0.3) 9 (1.3)
6 (2.9) 11 (5.3) 1 (0.5) 4 (1.9)
0 2 (2.4) 0 0
Haplotype nomenclature was italicized, as recommended by the HGVS. Haplotype frequencies are given as percentages in parentheses. n: number of chromosomes, RHD: rheumatic heart disease; RFo: rheumatic fever only. a Each haplotype block is separated by a space. First haplotype block include variants from the promoter to exon 3, second block include polymorphisms in intron 4 and 5, third block include variants in intron 9 and exon 10, fourth block include variants in exon 12. For nonsynonimous SNPs, aminoacid changes are indicated, e.g. CDV means a cytosine (C) at position 21081 in intron 9, followed by two closely located nucleotide substitutions encoding asparagine (D) at residue 371 and valine (V) at residue 377 of the protein. b In the alphanumerical system of the phylogenetic nomenclature, first clade is given by a number, followed by as many letters and numbers as branches/lineages in the tree [39]. In the case of MASP2, the system was added by a ‘‘l’’ for haplotypes generating ‘‘low’’ MASP-2 levels, ‘‘i’’ for haplotypes generating ‘‘intermediate’’ MASP-2 levels, ‘‘h’’ for haplotypes generating ‘‘high’’ MASP-2 levels [18]. It was also added by the alleles in introns 4 and 5 (within brackets), which are not in linkage disequilibrium with all other investigated SNPs [19]. Recombinant haplotypes are given by the names of the possible parental haplotypes, separated by a dot. Clade 1 haplotypes share g.24762C in exon 12: ⁄1A (most ancient), ⁄1B1-h and ⁄1B2-h (sharing g.21081T), ⁄1C1-l and ⁄1C2-l (sharing p.126L). Five other haplotypes present g.24762T in exon 12 and belong to clade 2: ⁄ 2A1, ⁄2A2-l (with the p.377A variant), ⁄2B1-I, the very common ⁄2B2A-i and ⁄2B2B-l (with the p.120G variant). c Reported effect of homozygous haplotypes [18,19]. d Reported results from in vitro studies. The effect of a recombinant protein with both p.126L and p.439H residues, encoded by ⁄1C2-l, is unknown, although a protein with p.439H is unable to bind MBL [17].
[AG]) increased the susceptibility to RHD among RF patients (22/206 or 10.7% in RHD patients vs. 2/84 or 2.4% in RFo patients, P = 0.02, OR = 4.9 [95% CI = 1.13–21.34]). This association remained after correction for age, which was the only demographic factor associated with RHD in comparison to RFo patients (P = 0.02).
and ethnic group: P < 0.0001, OR = 0.20 [95% CI = 0.09–0.47]) (Fig. 1). The difference remained after comparing controls and RHD, but not RFo patients, and was independent of sex, age and
3.2. MASP-2 levels Patients presented lower MASP-2 levels than controls, with medians 252.8 and 313.9 ng/ml, respectively (corrected for age
Fig. 1. MASP-2 levels in patients and controls. Medians and min–max ranges are shown. MASP-2 levels were compared with Mann–Whitney test.
Fig. 2. MASP-2 levels are associated with MASP2 genotypes. Medians and min–max ranges are shown. MASP-2 levels were compared with Kruskal–Wallis test. h: genotypes with haplotypes containing the suffix h (h/h and h/i), ii: homozygote genotypes with haplotypes containing the suffix i (i/i), l: genotypes with haplotypes containing the suffix l (l/l and l/i). We excluded 41 individuals (22 controls and 19 patients) due to ambiguous genotype–phenotype associations: seven were h/l heterozygotes and the others presented haplotypes not formerly associated with MASP-2 levels [18,19].
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ancestry (P < 0.0001, OR = 0.11 [95% CI = 0.03–0.42]). MASP-2 levels, nevertheless, did not differ between RHD and RFo patients (medians 254.9 and 241.6 ng/ml, respectively). We further confirmed previously reported associations of MASP2 haplotypes with MASP-2 levels, in both controls and patients. This was evident comparing genotypes with haplotypes reportedly associated with high MASP-2 concentrations (equal or higher than 600 ng/ml, produced by haplotypes containing the suffix h: h/h and h/i), intermediate concentrations (between 200 and 600 ng/ml, produced by haplotype with the suffix i: i/i) and low concentrations (less than or equal to 200 ng/ml, produced by haplotype with the suffix l: l/l and l/i). As expected, the genotype–phenotype association was more conspicuous in the control group (Fig. 2). Interestingly, we did not identify p.99Q among patients, which is a variant known to be associated with high MASP-2 levels (0.9% in controls, not significant) (Table 2).
4. Discussion Rheumatic fever is still a disease with great impact on the public health system of developing countries, where disease prevalence is high and expenses with cardiac surgeries, expressive [28]. Thus, attempts to elucidate the autoimmune and physiological mechanisms in this condition are important. Protection against invading pathogens relies on complex interactions between the genetically controlled innate and adaptive immune responses. In fact, several polymorphisms in genes that encode molecules involved in both innate and adaptive immune responses were shown to contribute to RF and RHD susceptibility [29]. The activation of complement cascade provides a first line of defense against Streptococcus pyogenes infections. Due to its importance in clearance of rheumatic etiological agents as well as in disposal of apoptotic bodies and potential autoimmune initiators, deficiencies of components of the lectin pathway have been found to increase susceptibility and modulate severity of most rheumatic disorders [30]. This is the first study to investigate a number of polymorphisms encompassing the whole MASP2 gene as well as related haplotypes and MASP-2 levels in patients with RF and RHD. We confirmed previously noticed associations between MASP2 polymorphisms/ haplotypes and MASP-2 levels [16–19] and the absence of an association with the p.D120G polymorphism [26,31]. MASP-2 levels were lower in patients, than in controls. These patients were the same formerly found with higher MBL levels [32]. Higher MBL and lower MASP-2 levels are consistent with MASP-2 consumption due to intense MBL-driven complement activation. Lower MASP-2 levels were also found in patients with myocardial infarction compared to controls, suggesting an involvement of the protein in complement activation following ischemia and myocardial necrosis [33]. On the other hand, higher MASP-2 levels were associated with improved survival in patients with hematologic malignancies, specifically lymphoma [34]. We further found an association of two SNPs (p.V377A and p.R439H) known to cause low MASP-2 levels [16,17], with protection against RF and RHD. p.R439H was reported also to protect against placental malaria [21]. In contrast, these SNPs were recently described to increase the susceptibility to leprosy [19]. The contrasting associations are not surprising, since MASP-2 modulates phagocytosis, complement and coagulation cascades, each exerting a different role in the susceptibility to infectious and autoimmune diseases [8,16,17]. Furthermore, the association between variants leading to low protein levels and protection against RF has been formerly found for MBL2 [35,36], but not for FCN2 [37]. The low basal concentrations of MBL and MASP-2 correspond to a lower capacity of complement activation, due to structural variants as p.52C, p.54D and p.57E in MBL2 and p.439H in MASP2 [30].
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This reinforces our suggestion that complement activation through the lectin pathway promote the inflammatory response and subsequent tissue damage in rheumatic fever disease. On the other hand, MASP2 CDVR haplotypes, encompassing g.1961795C, p.371D, p.377V and p.439R variants, where associated with an almost five times increased risk to RHD. They generate varying MASP-2 levels and are phylogenetically related to the most ancestral MASP2 haplotype [18]. These haplotypes probably harbor other polymorphisms not investigated in this study, which could contribute to the disease. Interestingly, the p.371D variant was also reported to increase susceptibility to HCV infection [20], D/D homozygotes were associated with bacterial infections after orthotopic liver transplantation [22] and the CD haplotype was found associated with susceptibility to chagasic cardiomyopathy [23]. Although cardiac commitments in Chagas disease and RHD cannot be compared, both may share autoimmune etiology [38]. In conclusion, the effects of MASP2 polymorphisms on protein serum levels and functional efficiency may modulate susceptibility to RF. MASP-2 levels were lower in patients, which may reflect protein consumption due to complement activation. This is in agreement with our former results regarding MBL protein and polymorphisms of the MBL2 gene. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD. Funding This work was supported by CNPq - Brazil (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES - Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Acknowledgments We gratefully acknowledge the patients for their participation in this study and thank the staff of the Laboratório de Imunopatologia Molecular HC/UFPR for their assistance. References [1] Chang C. Cutting edge issues in rheumatic fever. Clin Rev Allergy Immunol 2011:1–25. [2] Burke RJ, Chang C. Diagnostic criteria of acute rheumatic fever. Autoimmun Rev 2014;13:503–7. [3] Guilherme L, Kohler K, Kalil J. Rheumatic heart disease: mediation by complex immune events. Adv Clin Chem 2011;53:31–50. [4] Tibazarwa K, JA V, Mayosi B. Incidence of acute rheumatic fever in the world: a systematic review of population-based studies. Heart 2008;94:1534–40. [5] Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685–94. [6] Kumar RK, Tandon R. Rheumatic fever & rheumatic heart disease: the last 50 years. Indian J Med Res 2013;137:643–58. [7] Barbosa P, Muller R, Latado A, Achutti A, Ramos A, Weksler C. Diretrizes brasileiras para o diagnóstico, tratamento e prevenção da febre reumática. Arq Bras Cardiol 2009;93:1–18. [8] Mayilyan KR. Complement genetics, deficiencies, and disease associations. Protein Cell 2012;3:487–96. [9] Stäger S, Alexander J, Kirby AC, Botto M, Van Rooijen N, Smith DF, et al. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat Med 2003;9:1287–92. [10] Degn SE, Jensenius JC, Thiel S. Disease-causing mutations in genes of the complement system. Am J Hum Genet 2011;88:689–705. [11] Matsushita M, Endo Y, Fujita T. Structural and functional overview of the lectin complement pathway: its molecular basis and physiological implication. Arch Immunol Ther Exp (Warsz) 2013;61:273–83. [12] Stover CM, Thiel S, Thelen M, Lynch NJ, Vorup-Jensen T, Jensenius JC, et al. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene. J Immunol 1999;162:3481–90. [13] Thiel S. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins. Mol Immunol 2007;44:3875–88. [14] Laursen I, Thielens N, Christiansen M, Houen G. MASP interactions with plasma-derived MBL. Mol Immunol 2012;52:79–87.
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