Analysis for genotyping Duffy blood group in inhabitants of Sudan, the Fourth Cataract of the Nile

<p>Abstract</p> <p>Background</p> <p>Genetic polymophisms of the Duffy antigen receptor for the chemokines (DARC) gene successfully protected against blood stage infection by <it>Plasmodium vivax </it>infection. The Fy (a-, b-) phenotype is predominant among African populations, particularly those originating from West Africa, and it is rare among non-African populations. The aim of this study was to analyse the frequency of four Duffy blood groups based on SNPs (T-33C, G125A, G298A and C5411T) in two local tribes of Sudanese Arabs, the <it>Shagia </it>and <it>Manasir</it>, which are both from the region of the Fourth Nile cataract in Sudan.</p> <p>Methods</p> <p>An analysis of polymorphisms was performed on 217 individuals (126 representatives of the <it>Shagia </it>tribe and 91 of the <it>Manasir)</it>. Real-time PCR and TaqMan Genotyping Assays were used to study the prevalence of alleles and genotypes.</p> <p>Results</p> <p>The analysis of allelic and genotype frequency in the T-33C polymorphisms demonstrated a significant dominance of the <it>C </it>allele and <it>CC </it>genotype (OR = 0.53 [0.32-0.88]; p = 0.02) in both tribes. The G125A polymorphism is associated with phenotype Fy(a-, b-) and was identified in 83% of <it>Shagia </it>and 77% of <it>Manasir</it>. With regard to G298A polymorphisms, the genotype frequencies were different between the tribes (p = 0,002) and no single <it>AA </it>homozygote was found. Based on four SNPs examined, 20 combinations of genotypes for the <it>Shagia </it>and <it>Manasir </it>tribes were determined. The genotype <it>CC/AA/GG/CT </it>occurred most often in <it>Shagia </it>tribe (45.9%) but was rare in the <it>Manasir </it>tribe (6.6%) (p < 0.001 <it>Shagia </it>versus <it>Manasir</it>). The <it>FY*A^ES</it>allele was identified in both analysed tribes. The presence of individuals with the <it>FY*A/FY*A </it>genotype was demonstrated only in the <it>Shagia </it>tribe.</p> <p>Conclusion</p> <p>This is probably the first report showing genotypically Duffy-negative people who carry both <it>FY*B^ES</it>and <it>FY*A^ES</it>. The identification of the <it>FY*A^ES</it>allele in both tribes may be due to admixture of the non-African genetic background. Taken as a whole, allele and genotype frequencies between the <it>Shagia </it>and the <it>Manasir </it>were statistically different. However, the presence of individuals with the <it>FY*A/FY*A </it>genotype was demonstrated only in the <it>Shagia </it>tribe.</p

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fya/Fyb blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40–47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core

Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region

<p>Abstract</p> <p>Background</p> <p>Duffy blood group polymorphisms are important in areas where <it>Plasmodium vivax </it>predominates, because this molecule acts as a receptor for this protozoan. In the present study, Duffy blood group genotyping in <it>P. vivax </it>malaria patients from four different Brazilian endemic areas is reported, exploring significant associations between blood group variants and susceptibility or resistance to malaria.</p> <p>Methods</p> <p>The <it>P. vivax </it>identification was determined by non-genotypic and genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP in 330 blood donors and 312 malaria patients from four Brazilian Amazon areas. In order to assess the variables significance and to obtain independence among the proportions, the Fisher's exact test was used.</p> <p>Results</p> <p>The data show a high frequency of the <it>FYA/FYB </it>genotype, followed by <it>FYB/FYB, FYA/FYA</it>, <it>FYA/FYB-33 </it>and <it>FYB/FYB-33</it>. Low frequencies were detected for the <it>FYA/FY</it>^<it>X</it>, <it>FYB/FY</it>^<it>X</it>, <it>FYX/FY</it>^{<it>X </it>}and <it>FYB-33/FYB-33 </it>genotypes. Negative Duffy genotype (<it>FYB-33/FYB-33</it>) was found in both groups: individuals infected and non-infected (blood donors). No individual carried the <it>FY</it>^<it>X</it><it>/FYB-33 </it>genotype. Some of the Duffy genotypes frequencies showed significant differences between donors and malaria patients.</p> <p>Conclusion</p> <p>The obtained data suggest that individuals with the <it>FYA/FYB </it>genotype have higher susceptibility to malaria. The presence of the <it>FYB-33 </it>allele may be a selective advantage in the population, reducing the rate of infection by <it>P. vivax </it>in this region. Additional efforts may contribute to better elucidate the physiopathologic differences in this parasite/host relationship in regions endemic for <it>P. vivax </it>malaria, in particular the Brazilian Amazon region.</p

Vivax malaria in Mauritania includes infection of a Duffy-negative individual

<p>Abstract</p> <p>Background</p> <p>Duffy blood group polymorphisms are important in areas where <it>Plasmodium vivax </it>is present because this surface antigen is thought to act as a key receptor for this parasite. In the present study, Duffy blood group genotyping was performed in febrile uninfected and <it>P. vivax</it>-infected patients living in the city of Nouakchott, Mauritania.</p> <p>Methods</p> <p><it>Plasmodium vivax </it>was identified by real-time PCR. The Duffy blood group genotypes were determined by standard PCR followed by sequencing of the promoter region and exon 2 of the Duffy gene in 277 febrile individuals. Fisher's exact test was performed in order to assess the significance of variables.</p> <p>Results</p> <p>In the Moorish population, a high frequency of the <it>FYB^ES/FYB^ES</it>genotype was observed in uninfected individuals (27.8%), whereas no <it>P. vivax</it>-infected patient had this genotype. This was followed by a high level of <it>FYA/FYB</it>, <it>FYB/FYB</it>, <it>FYB/FYB^ES</it>and <it>FYA/FYB^ES</it>genotype frequencies, both in the <it>P. vivax</it>-infected and uninfected patients. In other ethnic groups (Poular, Soninke, Wolof), only the <it>FYB^ES/FYB^ES</it>genotype was found in uninfected patients, whereas the <it>FYA/FYB^ES</it>genotype was observed in two <it>P. vivax</it>-infected patients. In addition, one patient belonging to the Wolof ethnic group presented the <it>FYB^ES/FYB^ES</it>genotype and was infected by <it>P. vivax</it>.</p> <p>Conclusions</p> <p>This study presents the Duffy blood group polymorphisms in Nouakchott City and demonstrates that in Mauritania, <it>P. vivax </it>is able to infect Duffy-negative patients. Further studies are necessary to identify the process that enables this Duffy-independent <it>P. vivax </it>invasion of human red blood cells.</p

Duffy blood group genotypes among malaria patients in Rondônia, Western Brazilian Amazon Genótipos do sistema sanguíneo Duffy em pacientes maláricos de Rondônia, Amazônia Ocidental Brasileira

We have compared Duffy blood group genotype distribution, as determined by polymerase chain reaction with allele-specific primers, in 68 Plasmodium vivax-infected patients and 59 non-vivax malaria controls from Rondônia, Brazil. Homozygosity for the allele Fy, which abolishes Duffy antigen expression on erythrocytes, was observed in 12% non-vivax controls but in no P. vivax patient. However, no significant association was found between Fy heterozygosity and protection against P. vivax. The Fy x allele, which has recently been associated with very weak erythrocyte expression of Duffy antigen, was not found in local P. vivax patients.<br>Compara-se neste trabalho a distribuição de genótipos do sistema sangüíneo Duffy, determinados através de reação em cadeia da polimerase com oligonucleotídeos iniciadores alelo-específicos, em 68 pacientes com infecção por Plasmodium vivax e em 59 controles com malária não-vivax de Rondônia, Brasil. Nenhum paciente infectado com P. vivax, mas 12% dos controles não-vivax, eram homozigotos para o alelo Fy, que abole a expressão do antígeno Duffy em hemácias. No entanto, não se observou evidência de proteção significativa contra P. vivax entre indivíduos heterozigotos para Fy. O alelo Fy x, que tem sido recentemente associado com a expressão eritrocitária muito fraca do antígeno Duffy, não foi encontrado entre pacientes locais com infecção por P. vivax

Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development