The functional associations between il-4 -589t/c and Il-6 -636g/c promoter polymorphisms and paediatric Severe malarial anaemia in a holoendemic area of Western Kenya

Abstract

Plasmodium falciparum, the most virulent and lethal human malaria parasite, IS responsible for most malaria-related morbidity and mortality in sub-Saharan Africa. It manifests mainly as severe malarial anaemia (SMA) in children in malaria holoendemic areas such as western Kenya. So far, few studies have investigated genetic polymorphism in cytokines, which are important determinants of immune response regulation. Since previous studies have suggested that interleukin-4 (IL-4) and IL-6 production and their interactions seem to playa significant role in the pathogenesis of severe malaria, this study investigated the fuctional effects of IL-4 -589T/C and IL-6 - 636G/C gene promoter single nucleotide polymorphisms (SNPs) on IL-4 and IL-6 cytokine production and their associations with SMA and high-density parasitaemia (HDP) during malaria infections. This was a cross-sectional study based at Siaya District Hospital (SDH) and a total of 618 and 602 study participants were studied for IL-4 - 589T/C and IL-6 -636G/C SNPs, respectively. The study participants were grouped as aparasitaemic controls (AC), low density parasitaemia (LDP), HDP, SMA and nonsevere malarial anaemia (Non-SMA). This was after the study participants had met the inclusion criteria which considered their natural exposure to P. falciparum, prior hospitalisation, intended relocation, being P. falciparum positive or negative and signing of the consent forms by parents/guardians, amongst others. Three millilitres of venous blood samples were collected (in ethylene diamine tetracetic acid (EDT A) vacutainer tubes and tubes without anticoagulants) from aparasitaemic children and malaria parasitemic children presenting at SDH and were used for clinical, immunological, and molecular evaluations, including haemogl~ electrophoresis, genotyping, full haemogram and cytokine profile anal~es. The IL-4 -589T/C and IL-6 -636G/C genotyping was carried out using a Taqman S' allelic discrimination by real time PCR. Human Cytokine Twenty-Five Plex Antibody Bead Kit and a haematological analyzer were used to determine the cytokine levels and blood cell indices, respectively. The data were analysed by SPSS software. Kruskal Wallis test and the Marin-Whitney U tests were used for across group and pairwise comparisons, respectively. Proportionality was tested by X2 tests, while multivariate logistic regression analysis was used for determining the associations between the genotypes and malaria clinical outcomes, in a model controlling for the confounders (age, gender, sickle-cell trait, bacteremia and HIV status). Results showed that IL-4 -589T/C SNP was significantly associated with increased susceptibility to HDP (OR; 1.64, 95%CI; 1.01-2.65, P=0.044); however, the IL-6 - 636G/C SNP was not associated with any malaria disease outcome. In addition, the IL-4 -589T/C and IL-6 -636G/C SNPs were not functionally associated with circulating IL-4 and IL-6 levels, respectively. The significant departure from the Hardy-Weinberg equilibrium (HWE) shown by the IL-4 -589T/C and IL-6 -636G/C genotypic distributions, implicate the action of evolutionary forces on the genes, possibly through the exposure of the population to malaria. In conclusion, the variation in the IL-4 promoter region as found in this tudy seems to be conditioning the clinical outcomes of fa1ciparum malaria and as a result, should be useful in the development of novel interventions for the control and management of severe malaria. The results of IL-6 - 636G/C SNP from this study, .on the other hand; has shown that the polymorphism neither conditions malaria disease outcomes nor circulating IL-6 levels in this population, thus necessitating studies on additional IL-6 SNPs.