Apresentação do Programa de Avaliação Externa da Qualidade / INSA IP

<div><p><em>Plasmodium falciparum</em> malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of <em>FCGR2A</em> and exon 3 of <em>FCGR3B</em> genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. <em>FCGR3B</em>-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of <em>FCGR3B</em>-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: <em>p</em> = 0.0061; recessive: <em>p</em> = 0.097; dominant: <em>p</em> = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTG<b>A</b>AA) containing the 233A-allele (in bold) was associated with protection from malaria (<em>p</em> = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (<em>p</em> = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.</p> </div

<i>FCGR3B</i> allele frequencies and Hardy-Weinberg (HW) estimations in protected individuals.

*<p>Allele and amino acid numberings refer to positions in <i>FCGR3B</i> transcript ENST00000367964.</p>a<p>HW estimations based on children (n = 267) who had diploid copies of <i>FCGR3B</i>, were the first sibling in a family and were not susceptible to clinical malaria in the observation period.</p

Studied SNPs in the <i>FCGR3B</i> gene, linkage disequilibrium (LD) patterns and haplotype association analysis.

<p><b>A</b>) A schematic of exon 3 <i>FCGR3B</i> gene (<b>NM_000570.4</b>) and LD plot of the respective SNPs visualised using Haploview v4.2. The LD plot shows pairwise r² values (×100) given in the squares for each comparison between the SNPs. White squares represent r² values equal to 0. Different shades of grey represent r² values between 0 and 1. <b>B</b>) Haplotype associations with susceptibility to clinical malaria compared to clinically protected individuals. Odds ratio (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression controlling for age, gender, ethnicity, sickle-cell status, <i>FCGR3B</i> copy number, blood group and use of bed net. The haplotype with the highest frequency in the study population was considered the reference group in the multivariate logistic regression analyses. ^ŦVariant first reported in this study, the associated gene for NA1*06 is <i>FCGR3B*01A194G, G316A</i>; ^Ψ Variants first reported by Matsuo et al, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046197#pone.0046197-Matsuo1" target="_blank">[28]</a>, the associated genes for NA1*02, NA2*02 and NA2*03 are <i>FCGR3B*01G316A</i>, <i>FCGR3B*02G194A</i> and <i>FCGR3B*02A244G</i> respectively.</p

Allele frequencies and Hardy-Weinberg estimations in the CM and UM study populations.

a<p>values are statistically significant if <i>p</i><0.0125 (Bonferroni corrected significance threshold).</p

Single marker association of <i>FCGR3B</i> alleles with clinical malaria.

<p>Odds ratio (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression controlling for age, gender, ethnicity, sickle-cell status, <i>FCGR3B</i> copy number, blood group, family structure and use of bed net. MAF: minor allele frequency.</p>Ŧ<p>All individuals who never had malaria despite parasitaemia at any time point (monthly blood slide) during the study, plus all individuals who never had malaria but without detectable parasitaemia by microscopy.</p

rs5030738 (c.233C>A) allele distribution among malaria endemic and malaria non-endemic populations.

*<p>Allele frequency data from the 1000 Genomes project data base. AFR: African; AMR: Ad Mixed American; EUR: European.</p

<i>FCGR3B</i>-c.233C>A genotypes.

<p>Genotype distribution compared between malaria endemic (Ghanaian) and malaria non endemic (Danish) populations.</p

Covariates association with clinical malaria.

<p>Odds Ratios (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression.</p>a<p>Analysis for each covariate was adjusted by the other remaining covariates: age groups, sex, sickle cell status, blood group, bed net use and ethnic group. The likelihood ratio (LR) test result compares the adjusted model to a model which only includes the adjusting factors and thereby tests if the variable has an effect on susceptibility.</p

Single marker association with cerebral malaria and uncomplicated malaria.

<p>The frequency of the associated allele in uncomplicated malaria (UM) and CM is shown. Odds Ratios (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression controlling for age and gender. The reference groups in the multivariate logistic regression analyses were those without the respective alleles.</p

Map of the study area showing the dam and non-dam study communities.

<p>Credit: Mr. Richard Adade, GIS & Remote Sensing Unit, Department of Fisheries and Aquatic Sciences, Center for Coastal Management, University of Cape Coast, Cape Coast, Ghana.</p