Angelica Beate Winter Boldt

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan‐binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro–Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild‐type, and B, C or D also known as O) were evaluated using real‐time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme‐linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0·022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0·04) and to the control group (P = 0·011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non‐responsiveness to pginterferon and ribavirin treatment (P = 0·023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

MASP2 haplotypes are associated with high risk of cardiomyopathy in chronic Chagas disease

Mannose-binding lectin (MBL) initiates complement on Trypanosoma cruzi through the MBL-associated serine protease 2 (MASP2). We haplotyped six MASP2 polymorphisms in 208 chronic chagasic patients, being 81 indeterminate and 123 symptomatic (76 with cardiac, 19 with digestive and 28 with cardiodigestive forms) and 300 healthy individuals from Southern Brazil, using PCR with sequence-specific primers. The g.1961795C, p.371D diplotype (short CD) occurred at a higher frequency among symptomatic patients, compared with the indeterminate group (P(Bf)=0.012, OR=3.11), as well as genotypes with CD, but not with the g.1945560A in the promoter in cardiac patients (P(Bf)=0.012, OR=13.54). CD haplotypes linked to the p.P126L and p.V377A variants were associated with reduced MASP-2 levels (P<0.0001) but not reduced MBL/MASP-2/C4 complexes. MASP2*CD genotypes, most of them generating low MASP-2 levels, are associated with high risk of chagasic cardiomyopathy. Rapid MASP2 genotyping might be used to predict the risk of symptomatic disease.

Association of L-Ficolin Levels and FCN2 Genotypes with Chronic Chagas Disease

Background L-ficolin (encoded by FCN2) binds to acetylated sugar moieties of many pathogens, including Trypanosoma cruzi, promoting their phagocytosis and lysis by the complement system. Methods We investigated L-ficolin levels in 160 T. cruzi infected patients with chronic Chagas disease and 71 healthy individuals, and FCN2 polymorphisms (−986 G>A, −602 G>A, and −4 A>G in the promoter and A258S in exon 8) in 243 patients, being 88 indeterminate (asymptomatic), 96 with cardiac, 23 with digestive and 33 with cardiodigestive manifestations (two were unspecified) and 305 controls (135 for A258S). Results Patients presented lower L-ficolin plasma levels than controls (p<0.0001). Among the different groups of cardiac commitment, individuals with moderate forms had higher L-ficolin levels than the severe forms (P = 0.039). Lower L-ficolin levels were found associated with the 258S variant in the patients (P = 0.034). We found less −4A/G heterozygotes in the cardiac patients, than in the controls (OR = 0.56 [95% CI = 0.33–0.94], P = 0.034). Heterozygote −4A/G genotypes with the 258S variant and 258SS homozygotes were nevertheless more frequent among cardiodigestive patients than in controls (OR = 14.1 [95% CI = 3.5–56.8], P = 0.0001) and in indeterminate patients (OR = 3.2 [95% CI = 1.1–9.4], P = 0.037). We also found an association of the allelic frequency of the 258S variant with cardiodigestive Chagas disease compared to controls (OR = 2.24 [95% CI = 1.1–4.5], P = 0.037). Thus, decreased patient levels of L-ficolin reflect not only protein consumption due to the disease process, but also the higher frequency of the 258S variant in patients with cardiodigestive symptoms. Conclusion The very first study on Brazilian cohort associates both L-ficolin plasma levels and FCN2 variants to Chagas disease and subsequent disease progression. The prognostic value of L-ficolin levels and the FCN2*A258S polymorphism should be further evaluated in other settings.

MBL-associated serine proteases (MASPs) and infectious diseases.

Abstract The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases.

The Lectin Pathway of Complement and Rheumatic Heart Disease

The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever.

MASP2 gene polymorphism is associated with susceptibility to hepatitis C virus infection

Abstract Hepatitis C virus (HCV) has become a major public health issue and is prevalent in most countries. We examined several MASP2 functional polymorphisms in 104 Brazilian patients with moderate and severe chronic hepatitis C using the primers set to amplify the region encoding the first domain (CUB1), a critical region for the formation of functional mannan-binding lectin (MBL)/MBL-associated serine proteases (MASP)–2 complexes, and the fifth domain (CCP2), which is essential for C4 cleavage of the MASP2 gene. We identified five single nucleotide polymorphisms in patients and controls: p. R99Q, p. D120G, p.P126L, p.D371Y, and p.V377A. Our results show that the p.D371Y variant (c.1111 G > T) is associated with susceptibility to HCV infection (p = 0.003, odds ratio = 6.33, 95% confidence interval = 1.85–21.70). Considered as a dominant function for the T allele, this variant is associated with high plasma levels of the MASP-2 in hepatitis C patients (p < 0.001). However, further functional investigations are necessary to understand the degree of involvement between MASP2 and the HCV susceptibility.

Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19 serum levels.

Deficiency of mannan-binding lectin-associated serine protease 2 (MASP-2) has been associated with infections, whereas high levels appear to increase the risk of inflammatory disorders. Nevertheless, MASP2 haplotypes have been poorly investigated. To overcome haplotyping cost and time consumption, we developed multiplex polymerase chain reactions with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs), reducing the number of necessary reactions from 18 to 7. SNPs were distributed from the promoter to the last exon, and a single PCR-SSP was used for p.D120G. We evaluated the phylogenetic relationships and global distribution of 10 identified haplotypes in 338 Danish individuals with known MASP-2 and MAp19 levels and 309 South Brazilians. Four haplotypes were associated with reduced MASP-2 levels in plasma (lower than 200 ng/mL). Simultaneous association with the highest MASP-2 (over 600 ng/mL) and lowest MAp19 levels (lower than 200 ng/mL) was demonstrated with the intron 9 mutation (Kruskal-Wallis p < 0.0001). Cumulative genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both populations. Rapid and low-cost screening of patients with multiplex MASP2 PCR-SSP could be used to identify clinical conditions where MASP-2 (or MAp19) levels may be disease modifying, possibly improving disease outcome through early therapeutic and preventive measures.

Genetically Determined MBL Deficiency Is Associated with Protection against Chronic Cardiomyopathy in Chagas Disease

Chagas disease (CD) is caused by Trypanosoma cruzi, whose sugar moieties are recognized by mannan binding lectin (MBL), a soluble pattern-recognition molecule that activates the lectin pathway of complement. MBL levels and protein activity are affected by polymorphisms in the MBL2 gene. We sequenced the MBL2 promoter and exon 1 in 196 chronic CD patients and 202 controls. The MBL2*C allele, which causes MBL deficiency, was associated with protection against CD (P = 0.007, OR = 0.32). Compared with controls, genotypes with this allele were completely absent in patients with the cardiac form of the disease (P = 0.003). Furthermore, cardiac patients with genotypes causing MBL deficiency presented less heart damage (P = 0.003, OR = 0.23), compared with cardiac patients having the XA haplotype causing low MBL levels, but fully capable of activating complement (P = 0.005, OR = 7.07). Among the patients, those with alleles causing MBL deficiency presented lower levels of cytokines and chemokines possibly implicated in symptom development (IL9, p = 0.013; PDGFB, p = 0.036 and RANTES, p = 0.031). These findings suggest a protective effect of genetically determined MBL deficiency against the development and progression of chronic CD cardiomyopathy.

Leprosy association with low MASP-2 levels generated by MASP2 haplotypes and polymorphisms flanking MAp19 exon 5.

Background The gene MASP2 (mannan-binding lectin (MBL)-associated serine protease 2) encodes two proteins, MASP-2 and MAp19 (MBL-associated protein of 19 kDa), bound in plasma to MBL and ficolins. The binding of MBL/MASP-2 and ficolin/MASP-2 complexes to microorganisms activates the lectin pathway of complement and may increase the ingestion of intracellular pathogens such as Mycobacterium leprae. Methods We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 219 Brazilian leprosy patients (131 lepromatous, 29 borderline, 21 tuberculoid, 14 undetermined, 24 unspecified), 405 healthy Brazilians and 291 Danish blood donors with previously determined MASP-2 and MAp19 levels. We also evaluated MASP-2 levels in further 46 leprosy patients and 69 Brazilian controls. Results Two polymorphisms flanking exon 5 of MASP2 were associated with a dominant effect on high MASP-2 levels and an additive effect on low MAp19 levels. Patients presented lower MASP-2 levels (P = 0.0012) than controls. The frequency of the p.126L variant, associated with low MASP-2 levels (below 200 ng/mL), was higher in the patients (P = 0.0002, OR = 4.92), as was the frequency of genotypes with p.126L (P = 0.00006, OR = 5.96). The *1C2-l [AG] haplotype, which harbors p.126L and the deficiency-causing p.439H variant, has a dominant effect on the susceptibility to the disease (P = 0.007, OR = 4.15). Genotypes composed of the *2B1-i and/or *2B2A-i haplotypes, both associated with intermediate MASP-2 levels (200–600 ng/mL), were found to be protective against the disease (P = 0.0014, OR = 0.6). Low MASP-2 levels (P = 0.022), as well as corresponding genotypes with *1C2-l and/or *2A2-l but without *1B1-h or *1B2-h, were more frequent in the lepromatous than in other patients (P = 0.008, OR = 8.8). Conclusions In contrast with MBL, low MASP-2 levels increase the susceptibility to leprosy in general and to lepromatous leprosy in particular. MASP2 genotypes and MASP-2 levels might thus be of prognostic value for leprosy progression.

Association of MASP2 polymorphisms and protein levels with rheumatic fever and rheumatic heart disease.

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.