G6PD Polymorphisms and Hemolysis After Antimalarial Treatment With Low Single-Dose Primaquine: A Pooled Analysis of Six African Clinical Trials

Abstract

Primaquine (PQ) is an antimalarial drug with the potential to reduce malaria transmission due to its capacity to clear mature Plasmodium falciparum gametocytes in the human host. However, the large-scale roll-out of PQ has to be counterbalanced by the additional risk of drug-induced hemolysis in individuals suffering from Glucose-6-phospate dehydrogenase (G6PD) deficiency, a genetic condition determined by polymorphisms on the X-linked G6PD gene. Most studies on G6PD deficiency and PQ-associated hemolysis focused on the G6PD A- variant, a combination of the two single nucleotide changes G202A (rs1050828) and A376G (rs1050829), although other polymorphisms may play a role. In this study, we tested the association of 20 G6PD single nucleotide polymorphisms (SNPs) with hemolysis measured seven days after low single dose of PQ given at the dose of 0.1 mg/kg to 0.75 mg/kg in 957 individuals from 6 previously published clinical trials investigating the safety and efficacy of this drug spanning five African countries. After adjusting for inter-study effects, age, gender, baseline hemoglobin level, PQ dose, and parasitemia at screening, our analysis showed putative association signals from the common G6PD mutation, A376G [-log₁₀(p-value) = 2.44] and two less-known SNPs, rs2230037 [-log₁₀(p-value] = 2.60), and rs28470352 [-log₁₀(p-value) = 2.15]; A376G and rs2230037 were in very strong linkage disequilibrium with each other (R ² = 0.978). However, when the effects of these SNPs were included in the same regression model, the subsequent associations were in the borderline of statistical significance. In conclusion, whilst a role for the A- variant is well established, we did not observe an important additional role for other G6PD polymorphisms in determining post-treatment hemolysis in individuals treated with low single-dose PQ.

Keywords: clinical trials; drug safety; genetic association study; hemoglobin; malaria.

FIGURE 1

Inter-study variation of Hb0 and Hb7. (A) Violin plots of Hb0 per study. (B) Relationship between Hb0 and parasitemia in infected individuals. Note that the study from Mali is not shown because it only recruited non-infected individuals. (C) Violin plots of the ratio between Hb0 and Hb7.

FIGURE 2

Inter-study variation with respect to the genetic data. (A) Comparison of genotype frequencies between studies. Statistical significance was determined by –log₁₀ (p-value) associated with the Pearson’s test for independence in two-way frequency tables. For males and females, the thresholds for statistical significance were calculated separately and were determined to ensure a false discovery rate (FDR) of 5%. (B) Comparison between allelic frequencies between studies and 1000 Genome Project data. The reported –log₁₀ (p-value) refers to the minimum p-value of Binomial test for comparing the allelic frequency of a given SNP in each study assuming the respective allelic frequency of 1000G project data as the one under the null hypothesis (see Table 2).

FIGURE 3

Genetic association analysis between log-Hb7 and each SNP adjusting or not for parasitemia at day 0 (A,B, respectively). Statistical significance was determined by the maximum of −log₁₀ (p-value) associated with the log-likelihood ratio tests when testing different genetic effects of each SNP in a linear regression model adjusting for age, gender, parasitemia at screening, and log-Hb0. The threshold for statistical significance was determined to ensure a false discovery rate (FDR) of 5%.