Genetic variation in cytokines and effects as determinants of susceptibility and clinical outcomes of severe malarial anaemia in children in western Kenya
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Plasmodium falciparum malaria still affects 350-500 million people resulting in 0.7-2.7 million deaths annually. This cross-sectional study conducted in western Kenya focused on defining host genetic factors that influence clinical outcomes of severe malarial anemia (SMA) and hyperparasitaemia, the two most common clinical sequelae in children residing in this area. This hospital-based study focused on polymorphisms within the Fc gamma receptor Ila (FcyRIIA, CD32), Nitric oxide synthase type 2 (NOS2, INOS, G-954C) and Interleukin-1R -31 gene (IL-1(3 -31) and their roles in conditioning SMA and other related clinical manifestations in children below 3 years residing in this holoendemic P. falciparum transmission area of western Kenya. Clinical, demographic and parasitological evaluations were carried out on every study participant presenting to Siaya District Hospital with acute malaria. Beckman-Counter Counter TM and Giemsa-stained slides were used to determine complete blood counts and parasite density, respectively. DNA obtained from blood-blots was used for genetic analysis. PCR using gene-specific primers followed by allele-specific restriction fragment length polymorphism was used to genotype for different variants within each gene. For the functionality relationships between the NOS2 -954 and IL-l(3 -31 variants, the urinary nitrite (NO,) and circulating IL-1(3 levels were determined using Griess method and Human Cytokine Multiplex system, respectively. All statistical analyses were performed using SPSS and Minitab. Kruskal-Wallis tests and Chi-square analyses were used for group comparisons and proportions, respectively. Multivariate logistic regression, controlling for confounding factors was used to examine the association between genetic variants and malaria clinical outcomes. Analyses were performed using SMA (Hb < 6.0 g/dl) and high-density parasitaemia (HDP, parasites/µL≥ 10, 000). The reference group in these multivariate analyses was the wild-type genotype. Statistical significance was defined as p≤ 0.05. A total of 562, 556 and 569 individuals were genotyped for the FcyRIIA-131, NOS2 -954 and IL-1β -31 genes, respectively. For the FcyRIIA-131 gene, multivariate model- revealed that relative to HIR131, neither the RJR131 nor H/H131 genotypes were associated with protection against SMA or malarial anemia (MA). However, the R/R131 genotypes were significantly associated with protection against HDP (p = 0.02), while the H/11131 genotypes were mildly protective against HDP. At the NOS2 locus, multivariate logistic regression analysis revealed that relative to the GG genotypes, the CC group were significantly protected from MA (p = 0.04). Both CC and GC groups were mildly protected against HDP. Additionally, the CC genotype was associated with functionally higher production of NO levels relative to the other NOS2 genotypes. Further analyses at the ILlβ -31 gene revealed that relative to the CC genotype, the TT were significantly protected against SMA (p = 0.02) while the GC group were significantly susceptible to HDP (p 0.001). In addition, the TT genotypes were functionally associated with significantly higher IL-lβ production compared to the other genotypes (p < 0.05). Since there is currently no effective vaccine for malaria, the development of a vaccine that protects against SMA and HDP would be crucial in preventing the vast morbidity and mortality associated with the disease. In this study, some of the cytokines, effector and receptor mediators responsible for influencing SMA and HDP have been identified. These findings would provide useful information in the development and testing of an effective malaria vaccine in children from western Kenya, a holoendemic P. falciparum transmission area.