Host Genetic Factors in Glucose-6-Phosphate Dehydrogenase and Cytochrome B5 Reductase 3 Affect the Susceptibility of Developing Severe Malarial Anemia

Abstract

Abstract Host genetic factors that influence the outcome of Plasmodium falciparum malaria infection are not fully understood. Glucose-6-phosphate dehydrogenase (G6PD), an X-linked gene, encodes the sole enzyme in red blood cells that produces NADPH for protection from reactive oxygen species. G6PD A+ (G6PD c. 376G) is an African specific polymorphism reported to have reduced activity1 but no apparent phenotype; G6PD A- (G6PD c 202A/376G) is a related polymorphism with decreased activity and increased risk for oxidant-induced hemolysis2. Previous investigators have reported that G6PD deficiency provides a protective effect from malaria3. A recent Malaria Genomic Epidemiology Network (MalariaGEN) study with almost 30,000 participants reported that G6PD A- increases the risk for severe malarial anemia4. Cytochrome b5 reductase 3 (CYB5R3) in red blood cells transfers electrons from NADH to cytochrome b5, which in turn converts methemoglobin to hemoglobin. The CYB5R3 T117S variant, an African-specific polymorphism with a prevalence higher than previously described African-specific polymorphisms (allele frequency .23)5, is not associated with methemoglobinemia. We hypothesized that CYB5R3 T117S may protect from severe malarial anemia, possibly by enhanced anti-oxidative potential of erythrocytes through higher NADH levels. We isolated DNA from dried blood spots from 133 children (age < 6 years) who presented to hospital in southern Zambia with clinical malaria. Sixty-seven had severe anemia (hematocrit <15%) and 66 had uncomplicated malaria (hematocrit ≥18%); all had normal coma scores. We determined G6PD A+, G6PD A- and CYB5R3 T117S by Taqman genotyping. We also isolated DNA from plasma samples and genotyped for CYB5R3 T117S. There was 97.7% agreement in the genotyping. The overall prevalence of G6PD A+ was 20.3% and of G6PD A- 12.0%. The gene frequency of CYB5R3 T117S was .31. We examined the association of these genotypes with severe malarial anemia in logistic regression models that adjusted for body weight, duration of febrile illness before presentation, and treatment with traditional herbal medicine or sulfadoxine-pyrimethamine before presentation6. In keeping with the MalariaGEN study, we found that G6PD A- increased the odds of severe malarial anemia (OR 8.2; 95% CI 1.6-42.7l; P=0.013), but we also observed a trend with G6PD A+ (OR 2.1, 95% CI 0.7-6.5; P=0.22). We therefore assessed the additive effect of these polymorphisms and observed a progressive increase in the risk with G6PD A+ and G6PD A- (OR 2.6, 95% CI 1.3-5.3). We added CYB5R3 T117S to this model and found a non-significant trend to a progressive reduction in the risk of severe anemia with heterozygosity and homozygosity for T117S (OR = 0.7, 95% CI 0.3-1.4; P=0.29). In further analysis, we observed an interaction between CYB5R3 T117S and G6PD genotype in the risk for severe anemia (P =0.092). We therefore stratified our analysis according to the presence or absence of G6PD variants. In the absence of G6PD A+ or A-, CYB5R3T117S offered protection against severe anemia (OR 0.3, 95% CI 0.1-0.9, P=0.035) in an additive model. In contrast, in the presence of G6PD A+ or G6PDA-, CYB5R3 T117S mutation tended to increase the odds of severe anemia in malaria (OR 3.1, 95% CI 0.6-15.9, P=0.18). In summary, 1) we confirm the association of G6PD A- with severe malarial anemia in southern Zambian children, 2) we observe an additive increased risk of severe malarial anemia with G6PD A+ and G6PD A-, and 3) we report heterogeneity of the effect of CYB5R3 T117S on the risk of severe anemia according to G6PD A+ and A- status. The observations with CYB5R3 T117S need to be confirmed in a larger cohort and the underlying mechanisms worked out through laboratory and translational research. We conclude that the combined effect of host genetic factors in two different red cell redox regulating enzymes may affect the outcome of P. falciparum infection. ReferencesGomez-Manzo, S. et al. International journal of molecular sciences16, 28657-28668 (2015).Luzzatto, L., Nannelli, C. & Notaro, R. Hematology/oncology clinics of North America30, 373-393 (2016).Ruwende, C. et al. Nature376, 246-249 (1995).Rockett, K. A. et al. Nature genetics46, 1197-1204 (2014).Jenkins, M. M. & Prchal, J. T. Hum Genet99, 248-250 (1997).Thuma, P. E. et al. J Infect Dis203, 211-219 (2011). Disclosures Thuma: Malaria Institute at Macha: Employment.