Angelica Bw Boldt

Association of a new mannose-binding lectin variant with severe malaria in Gabonese children

Mannose-binding lectin (MBL2) variants that decrease the plasma level of the protein or encode dysfunctional proteins are frequently associated with the severity of a number of infections and autoimmune disorders. The high frequencies of these variants in most populations of the world are probably maintained by some selective advantage against widespread diseases. We found 14 new MBL2 allelic haplotypes, two of them with non-synonymous variants, by screening 136 children with uncomplicated malaria, 131 children with severe malaria and 39 older healthy schoolchildren. We also found a significant association of a novel variant with susceptibility to severe malaria (P=0.010). Increased MBL plasma levels and corresponding MBL2 genotypes were associated with lower concentration of several cytokines and chemokines in plasma of malaria patients. We suggest that malaria could have been one of the evolutionary driving forces shaping the MBL2 polymorphism in the African population.

The Association between Mannan-Binding Lectin Gene Polymorphism and Clinical Leprosy: New Insight into an Old Paradigm

BACKGROUND Mannan-binding lectin (MBL), a soluble protein of innate immunity, is known to play a role in pathogen recognition and clearance. For more than a decade, it has been proposed that MBL deficiency may be protective against intracellular pathogens, such as Mycobacterium leprae. METHODS The polymorphisms at the promoter and exon 1 regions of the MBL2 gene were assessed by polymerase chain reaction and sequencing performed on 264 patients with leprosy and 214 matched healthy control subjects from southern Brazil. RESULTS. The distribution of MBL2-gene polymorphisms in patients was significantly different from that in controls, with a decreased frequency of haplotypes/genotypes associated with low expression of circulating MBL in lepromatous patients when compared with tuberculoid patients (odds ratio [OR] for haplotypes, 0.56 [95% confidence interval {CI}, 0.33-0.93] [P=.020]; OR for genotypes, 0.31 [95% CI, 0.13-0.71] [P=.004]). The LYPA haplotype was associated with susceptibility to leprosy per se (OR, 2.25 [95% CI, 1.31-3.88] [P=.003]) and to progression to the lepromatous (OR, 2.2 [95% CI, 1.21-4.05] [P=.008]) and borderline (OR, 2.98 [95% CI, 1.29-6.87] [P=.008]) forms of the disease. CONCLUSIONS These results suggest that MBL2-gene polymorphisms play a role in susceptibility to leprosy per se and in the clinical progression of the disease.

Phylogenetic nomenclature and evolution of mannose-binding lectin (MBL2) haplotypes

BackgroundPolymorphisms of the mannose-binding lectin gene (MBL2) affect the concentration and functional efficiency of the protein. We recently used haplotype-specific sequencing to identify 23 MBL2 haplotypes, associated with enhanced susceptibility to several diseases.ResultsIn this work, we applied the same method in 288 and 470 chromosomes from Gabonese and European adults, respectively, and found three new haplotypes in the last group. We propose a phylogenetic nomenclature to standardize MBL2 studies and found two major phylogenetic branches due to six strongly linked polymorphisms associated with high MBL production. They presented high Fst values and were imbedded in regions with high nucleotide diversity and significant Tajimas D values. Compared to others using small sample sizes and unphased genotypic data, we found differences in haplotyping, frequency estimation, Fu and Lis D* and Fst results.ConclusionUsing extensive testing for selective neutrality, we confirmed that stochastic evolutionary factors have had a major role in shaping this polymorphic gene worldwide.

Association of deficiency for mannan-binding lectin with anti-mannan antibodies in Crohn's disease: A family study

Background: Antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall, are more frequently found in Crohns disease (CD) patients with low levels of mannan‐binding lectin (MBL). MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL is related to ASCA production in healthy family members of CD patients. Methods: ASCA and MBL concentrations in sera from patients (n = 52), and their 158 healthy relatives were measured by enzyme‐linked immunosorbent assay (ELISA). Genetic MBL variants were determined by DNA sequencing. Results: Thirty‐five (67%) patients were ASCA‐positive. Twenty‐six (74%) of the 35 ASCA‐positive patients had low MBL levels (<500 ng/mL), whereas only 4 (24%) of the 17 ASCA‐negative patients had low values for MBL (P = 0.001). ASCA were found in 38 (24%) family members. Twenty‐three (50%) of 46 family members with low values for MBL were ASCA‐positive compared to 15 (13%) of 112 family members with normal values for MBL (P < 0.0001). ASCA were found in 33 of 104 (32%) family members of ASCA‐positive patients and in 5 family members (9%) of ASCA‐negative patients (P = 0.002). Relatives with mutations leading to MBL deficiency had significantly more frequent ASCA than relatives without these mutations (P = 0.018). Conclusions: MBL deficiency is associated with ASCA positivity not only in patients with CD, but also in their relatives. An impaired innate immune system defined by low MBL serum concentrations may lead to an increased reactivity of the specific immune system to mannan antigens, and therefore facilitate the generation of ASCA.

Association of two variants of the interferon-alpha receptor-1 gene with the presentation of hepatitis B virus infection

Interferon-alpha (IFNalpha) is a critical mediator of immunity to hepatitis B virus (HBV) infection. Although IFN has been used in the treatment of viral hepatitis for more than a decade, the role of IFN-alpha-receptor in HBV infection has not been intensively studied. We have evaluated the impact of two variants of the IFNAR1 gene on the outcome of HBV infection. Four hundred and fifty eight HBV-infected Vietnamese patients, with well-characterised clinical profiles including all forms of hepatic disease, and 160 non-infected, healthy Vietnamese individuals were enrolled in the study. Of these patients, 54 had acute hepatitis B, 88 had chronic hepatitis B, 118 had liver cirrhosis, 146 had a hepatocellular carcinoma and 52 were asymptomatic carriers of HBV. We analysed two SNPs for unequal distribution between these groups. The first SNP, rs1012335 is situated in intron 3 of the interferon alpha receptor 1 (IFNAR1). A C at position 17470 in the IFNAR1 on both chromosomes was detected more frequently in HBV-infected patients compared to healthy controls (OR: 2.6; 95% CI: 1.46-4.72, p < 0.001). The same homozygosity is also associated with higher concentrations of AST and ALT (aspartate and alanine amino-transferase) in the plasma of the patients. The second SNP (rs2257167) is situated in exon 4, causing a change of amino acids from Val (GTT) to Leu (CTT). Subjects having GTT on both chromosomes were more frequent in the healthy control group (OR: 0.54, 95% CI: 0.35-0.84, p = 0.004) and had lower plasma ALT concentrations. The findings indicate that two variants of the IFNAR1 gene are associated with the clinical presentation of HBV infection.

Haplotype specific-sequencing reveals MBL2 association with asymptomatic Plasmodium falciparum infection

BackgroundMannose binding lectin (MBL) has an important role in the activation of the complement system and opsonization of pathogenic microorganisms. Frequent polymorphisms found in the MBL2 gene affect the concentration and functionality of the protein and are associated with enhanced susceptibility to severe malaria in African children. Most MBL2 typing strategies were designed to the analysis of selected variants, the significance of whole haplotypes is poorly known. In this work, a new typing strategy was developed and validated in an association analysis of MBL2 with adult asymptomatic infection.MethodsMBL2 allele-specific fragments of 144 healthy Gabonese adults were amplified by using haplotype-specific sequencing (HSS), a new strategy that combines sequence-specific PCR and sequence-based typing. The Gabonese were investigated for the presence of Plasmodium falciparum parasitaemia by the amplification of parasite genes, immunochromatographic antigen detection and microscopic analysis. HSS results were also compared with a previously used real-time PCR (RT-PCR) method in 72 Euro-Brazilians.ResultsFourteen polymorphisms were identified beside the commonly investigated promoter (H, L; X, Y; P, Q) and exon 1 (A, O; O = B, C or D) variants. The MBL2*LYPA/LYPA genotype was associated with the absence of asymptomatic infection (P = 0.017), whereas the MBL2*LYQC haplotype and YA/YO + YO/YO genotypes were associated with positive parasite counts in asymptomatic adults (P = 0.033 and 0.018, respectively). The associations were specific to LYPA (identical to the reference sequence Y16577) and LYQC (Y16578) and would not have been revealed by standard genotyping, as there was no association with LYPA and LYQC haplotypes carrying new polymorphisms defined by sequence-based typing. In contrast, HSS and RT-PCR produced very similar results in the less diverse European-derived population.ConclusionIn this work, a new typing strategy for a highly polymorphic gene was developed and validated focusing on the asymptomatic status of P. falciparum- infected adults. In populations with high nucleotide diversity, it allowed for the identification of associations with fine-scaled haplotypes that would not have been found using common typing techniques. In this preliminary study, MBL2 haplotypes or SNPs linked to them were found associated with susceptibility to infection and parasitaemia control of asymptomatic adults.

Analysis of the CCR5 gene coding region diversity in five South American populations reveals two new non-synonymous alleles in Amerindians and high CCR5*D32 frequency in Euro-Brazilians

The CC chemokine receptor 5 (CCR5) molecule is an important co-receptor for HIV. The effect of the CCR5*D32 allele in susceptibility to HIV infection and AIDS disease is well known. Other alleles than CCR5*D32 have not been analysed before, neither in Amerindians nor in the majority of the populations all over the world. We investigated the distribution of the CCR5 coding region alleles in South Brazil and noticed a high CCR5*D32 frequency in the Euro-Brazilian population of the Paraná State (9.3%), which is the highest thus far reported for Latin America. The D32 frequency is even higher among the Euro-Brazilian Mennonites (14.2%). This allele is uncommon in Afro-Brazilians (2.0%), rare in the Guarani Amerindians (0.4%) and absent in the Kaingang Amerindians and the Oriental-Brazilians. R223Q is common in the Oriental-Brazilians (7.7%) and R60S in the Afro-Brazilians (5.0%). A29S and L55Q present an impaired response to β-chemokines and occurred in Afro- and Euro-Brazilians with cumulative frequencies of 4.4% and 2.7%, respectively. Two new non-synonymous alleles were found in Amerindians: C323F (g.3729G > T) in Guarani (1.4%) and Y68C (g.2964A > G) in Kaingang (10.3%). The functional characteristics of these alleles should be defined and considered in epidemiological investigations about HIV-1 infection and AIDS incidence in Amerindian populations.

Variant alleles of the mannose binding lectin 2 gene (MBL2) confer heterozygote advantage within Crohn's families.

TO THE EDITOR: Mannose binding lectin (MBL) is a pattern recognition molecule of the innate immune system. Single nucleotide polymorphisms (SNPs) in the proximal promoter (L/H and X/Y), the 5¢UTR (P/Q) and SNPs in exon 1 at codons 52 (MBL2*D), 54 (MBL2*B), and 57 (MBL2*C) account for alterations in complement activation and decreased levels of MBL [1]. Researchers have reported the contribution of variant MBL alleles to disease susceptibility in infancy [2], spontaneous abortions, miscarriages and low weight newborns [3]. Other studies contemplated that the high frequency of these alleles is promoted by selective advantages providing protection against infections [4]. To evaluate the heterozygote advantage hypothesis in Crohn’s disease (CD), we analyzed individuals from 37 families. The cohort comprised 138 Caucasian individuals, among them 115 individuals were unaffected and 23 individuals had CD. Fifty-eight were parents (31 women, 27 men) and 65 were children (37 male and 28 female). Allelic variants of the MBL2 gene from individuals of families were determined by sequencing its promoter region and exon 1 [5]. Haplotype frequencies were estimated and a robust transmission disequilibrium test (rTDT) was performed to compare transmission of each of the two alleles of six SNPs [6]. Since there was no association of MBL haplotypes with CD, the promoter variants were ignored and all MBL variant alleles were pooled, i.e. SNPs in exon 1 as O haplotype and normal MBL2 allele as A haplotype. The segregation pattern was inferred across A/A, A/O and for O/O haplotypes. A Fisher’s exact test on these patterns was performed to validate whether all allelic combinations (A/A, A/O and O/O) contribute to CD. MBL2 genotype frequencies do not differ significantly between individuals affected and unaffected by Crohn’s disease (p = 0.928). The rTDT analysis revealed no significant preferential transmission of the alleles and haplotypes of the SNPs (Table I). Validation of all allelic combinations (A/A, A/O and O/O) remained insignificant (p = 0.195). In addition, neither the wild type (A/A, A/O) nor the mutant (O/O) contributed to disease (p = 0.324) (Table II). In accordance with other studies in Caucasians, HYPA and LXPA remained the most prominent haplotypes followed by LYPB, HYPD and LYQA [7]. Since no significant preferential transmission of the alleles or the haplotypes of the SNPs across generations was inferred, this led us to theorize the unknown advantage of these MBL2 variants and evaluate whether they confer any vital advantage on carrier generation. None of the observed frequencies of variants deviated from the expected. Studies have proposed that the complexity of signatures of possible

Reduced CD3/TCR complex expression leads to immunosuppression during Plasmodium falciparum malaria

Inhibition of T cell function is an important pathological feature in malaria. We investigated which T cell population is reduced contributing to immunosuppression. We examined protein and RNA level of various cell receptors, specific for T cells in children having Plasmodium falciparum infection and compared those to healthy children of the same age. We observe reduced levels of cluster of differentiation (CD)3 and T cell receptor (TCR)αβ in both RNA and protein expression level. This reduced expression was associated with a collapsed membrane asymmetry as determined by enhanced annexinV binding. Also human leukocyte antigen (HLA)-A,B,C- and HLA-DR-positive cells increasingly bound annexinV. The enhanced binding of annexinV was paralleled by a reduced expression of transcription factors such as T cell transcription factor 7 and GATA binding protein 3, which are involved in the expression of T cell specific genes. Also the expression of the transcription factors major histocompatibility complex class II transactivator and regulatory factor X 5, which are part of the HLA transcription machinery, is reduced during infection. We show that two mechanisms may lead to a suppression of the immune system during malaria: cell damage and reduction of gene expression of the CD3/TCR complex.