The CYB5R3c .350C>G and G6PD A alleles modify severity of anemia in malaria and sickle cell disease

Abstract

Genetic modifiers of anemia in Plasmodium falciparum infection and sickle cell disease (SCD) are not fully known. Both conditions are associated with oxidative stress, hemolysis and anemia. The CYB5R3 gene encodes cytochrome b5 reductase 3, which converts methemoglobin to hemoglobin through oxidation of NADH. CYB5R3^{c.350C > G} encoding CYB5R3^T117S , the most frequent recognized African-specific polymorphism, does not have known functional significance, but its high allele frequency (23% in African Americans) suggests a selection advantage. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection from oxidants; its African-polymorphic X-linked A+ and A- alleles, and other variants with reduced activity, coincide with endemic malaria distribution, suggesting protection from lethal infection. We examined the association of CYB5R3^{c.350C > G} with severe anemia (hemoglobin <5 g/dL) in the context of G6PD A+ and A- status among 165 Zambian children with malaria. CYB5R3^{c.350C > G} offered protection against severe malarial anemia in children without G6PD deficiency (G6PD wild type or A+/A- heterozygotes) (odds ratio 0.29, P = .022) but not in G6PD A+ or A- hemizygotes/homozygotes. We also examined the relationship of CYB5R3^{c.350C > G} with hemoglobin concentration among 267 children and 321 adults and adolescents with SCD in the US and UK and found higher hemoglobin in SCD patients without G6PD deficiency (β = 0.29, P = .022 children; β = 0.33, P = .004 adults). Functional studies in SCD erythrocytes revealed mildly lower activity of native CYB5R3^T117S compared to wildtype CYB5R3 and higher NADH/NAD+ ratios. In conclusion, CYB5R3^{c.350C > G} appears to ameliorate anemia severity in malaria and SCD patients without G6PD deficiency, possibly accounting for CYB5R3^{c.350C > G} selection and its high prevalence.

FIGURE 1

Zambian early childhood malaria cohort. There is a progressive increase in the proportion of children with severe anemia in the two risk categories of G6PD A alleles: (1) hemizygosity or homozygosity for G6PD A+ and (2) hemizygosity or homozygosity for G6PD A− (P = .003 after adjustment for weight-for-age z-score, splenomegaly, stool parasites and plasma concentrations of interleukin-10 and tumor necrosis factor-alpha, the most significant independent predictors of severe malarial anemia in this cohort as we previously reported)

FIGURE 2

Zambian early childhood malaria cohort. We observed an interaction between CYB5R3^{c.350C > G} genotypes (wildtype, heterozygote, hemizygote) and risk categories of G6PD A alleles on severe anemia (P = .017). We therefore stratified our analysis according to the presence or absence of G6PD risk categories. A, In G6PD wildtype children or heterozygotes for A alleles, CYB5R3^{c.350C > G} offered protection against severe anemia (P = .030 after adjustment for the covariates mentioned in Figure 1). B, In hemizygotes or homozyogtes for G6PD A+ or G6PD A−, CYB5R3^{c.350C > G} did not show protection from severe malarial anemia

FIGURE 3

Children and adolescents with SCD. A, The adjusted hemoglobin concentration is higher in PUSH CYB5R3^{c.350C > G} heterozygotes and homozygotes who possess G6PD wildtype alleles or are heterozygotes for G6PD A alleles. B, This does not apply to those who are hemizygotes or homozygotes for G6PD A+ or A−. C, The adjusted hemolytic component (derived by principal components analysis from reticulocytes and serum LDH, AST and bilirubin concentrations) is progressively lower in CYB5R3^{c.350C > G} heterozygotes and homozygotes who are G6PD wildtype subjects or heterozygotes for A alleles. D, This does not apply to those who are G6PD A+ or A− hemizygotes or homozygotes

FIGURE 4

Walk-PHaSST adults and adolescents with SCD. A, The adjusted hemoglobin concentration is progressively higher in the CYB5R3^{c.350C > G} heterozygotes and homozygotes compared to CYB5R3 wildtype subjects B, but not in the hemizygotes or homozygotes for G6PD A+ or A−. C, The hemolytic component is progressively lower in the CYB5R3^{c.350C > G} heterozygotes and homozygotes compared to CYB5R3 wildtype subjects both in subjects who are G6PD wildtype or G6PD A allele heterozygotes and D, among those who are hemizygotes or homozygotes for G6PD A+ or A−

FIGURE 5

There is a progressive decrease in the erythrocyte CYB5R3 enzymatic activity in CYB5R3^{c.350C > G} heterozygotes and homozygotes compared to wildtype in SCD patients. Shown are geometric mean and SE activity after adjustment for laboratory where assay was performed. Walk-PHaSST 40 patients, ARUP 31 patients, UIC 25 patients

FIGURE 6

In multiple linear regression analysis, NADH/NAD+ ratio increases with CYB5R3^{c.350C > G} genotype in 19 UIC SCD patients. Shown are mean and SE of the NADH/NAD+ ratio as adjusted for G6PD A+ and A−