In rural villages of Mali, where virtually all young children experience episodes of malaria, protective hemoglobinopathies and erythrocyte polymorphisms offer a tremendous survival benefit when they prevent progression of uncomplicated malaria to severe, life-threatening disease. Our study was designed to test for protection by the A− form of G6PD deficiency and determine whether the effect of this sex-linked polymorphism would be more evident in male or female children. Since children who never contract malaria are rare at our study sites, we used case-control comparisons of severe malaria patient “cases” against uncomplicated malaria patient “controls” as described by Hayes et al. [20
] for epidemiological assessments of protection.
Our results from the Malinké and Dogon children in two regions 600 km apart show that G6PD A− provides substantial protection against severe malaria in hemizygous males but little or no protection in heterozygous females. After correcting for the confounding variables of age-dependent acquired immunity and potential effects of hemoglobins C and S, we calculate a pooled OR of 0.28 (95% CI 0.11–0.62) for the males 5 y old and below in these two groups. While our case-control design does not allow the calculation of relative risk that would be available from a prospective cohort study, the nearly complete recruitment of children with malaria in the Kela region of our study suggests roughly two-thirds less risk of severe malaria in young G6PD-deficient relative to G6PD-normal males. This level of protection from G6PD A− in hemizygous males 5 y old and younger is comparable to levels reported for hemoglobins C and S in certain West African populations [13
Because homozygous G6PD-deficient females are relatively rare, the number of these individuals that could be recruited to our studies was low. Nevertheless, cohort sizes from the Kela region were sufficiently large to detect a possible trend to protection against severe malaria in these individuals. For female children 5 y old and younger, the age range benefiting most from this effect, the OR of this trend was calculated to be 0.24 (95% CI 0.01–1.63), a value comparable to that for young hemizygous males. Pooled OR calculations including homozygous females 5 y old and younger from additional regions were not possible as sufficient recruitment numbers were not available. Additional studies with larger groups will be necessary to confirm the significance of this trend in young homozygous females.
Two other studies have reported clinical investigations of severe malaria in relation to G6PD A−. In Nigeria, Gilles et al. [6
] observed a protective effect of G6PD A− against convulsions or coma in male children 6 mo to 4 y of age; numbers from the female cases were not considered statistically significant for conclusions. In a later and much larger study from The Gambia and Kenya, Ruwende et al. [5
] concluded that G6PD A− is associated with a 46%–58% reduction in the risk of severe malaria for both hemizygous males and heterozygous females. Those conclusions are different from ours and led us to look more deeply into the details of the authors' data and analysis.
Our first comment on the different conclusions from our study and that of Ruwende et al. [5
] concerns the control groups. Ruwende et al.
] compared cases of uncomplicated or severe malaria in children up to 10 y of age against age- and location-matched control children who did not have malaria. Controls in Kenya included healthy community children with or without asymptomatic parasitemia; controls in The Gambia included inpatient and outpatient children with nonmalarial illnesses and no P. falciparum
parasitemia. Comparative assessments of G6PD deficiency in cases of severe versus uncomplicated malaria were not reported. In contrast, our study was performed over two or four transmission seasons in Malian villages where nearly every child experienced malaria. Malaria-free “control” groups in this context would have been highly skewed with confounding variables if not impossible. Additionally, subjective thresholds of treatment-seeking behavior for uncomplicated malaria can vary among different village settings. We therefore focused on the natural selection of G6PD deficiency that arises from its ability to protect children against progression from uncomplicated to severe, life-threatening malaria.
Ruwende et al. [5
] provided data from The Gambia and Kenya on the prevalence of G6PD A− in children with severe or uncomplicated malaria. Using these data in stratified analysis, we calculate a pooled OR of 0.68 (95% CI 0.33–1.39; p
= 0.32) for the protective effect of G6PD A− in the hemizygous males, whereas for the heterozygous females the pooled OR is 0.91 (95% CI 0.55–1.53; p
= 0.71) (Table S1
). Inclusion in these data of children older than 5 y and children with Hb types other than AA could have masked a greater trend to protection in the hemizygous males. The numbers of recruited homozygous female cases were few and showed no suggestion of protection.
Taken together, what do the different clinical studies of severe malaria in relation to the G6PD A− form of deficiency indicate for an overall estimate of relative protection? In the investigations producing the data from the Dogon, Malinké, Kenyan, and Gambian populations, the study designs, definitions of severe and uncomplicated malaria, and methodological details were similar in their significant aspects and are, therefore, informative when analyzed together. Earlier data from the Nigerian study [6
] have to be excluded because of that study's highly restrictive definition of severe malaria (only cases of convulsions or coma in the presence of high fever and parasitemia ≥100,000/μl). We therefore performed fixed effects meta-analysis on the data from Ruwende et al. [5
] and from all children in our study (data from , not , to avoid selective exclusion of cases in this combined treatment). Pooled ORs from all four sets of data confirmed highly significant protection for hemizygous males (OR = 0.51; 95% CI 0.33–0.77; p
< 0.001), but not for heterozygous females (OR = 0.96; 95% CI 0.68–1.34; p
= 0.87). This overall indication of protection is probably underestimated for hemizygous HbAA males who are 5 y old and younger, in light of our findings that G6PD deficiency reduces the risk of severe malaria more in this population.
We also performed meta-analysis of our data and those of Ruwende et al.
] for evidence of protection against two leading forms of severe malaria highly fatal to African children, cerebral malaria and severe anemia. Greater numbers of cases of cerebral malaria were present in the data than cases of severe anemia (Table S2
). Results from this analysis showed statistically significant protection against cerebral malaria for hemizygous males (pooled OR = 0.44; 95% CI 0.24–0.77; p
= 0.002) but not for heterozygous females (pooled OR = 0.91; 95% CI 0.59–1.39; p
= 0.76). No significant OR for protection could be demonstrated for the recruitment groups with severe anemia (Table S2
G6PD deficiency provides an important example of a sex-linked locus that can be maintained in the absence of heterozygote advantage [21
]. Haplotype diversity and linkage disequilibrium analysis has indicated that the G6PD
*A− allele arose within the past 3,840–11,760 years and spread with agriculture and malaria in Africa [22
]. It is a balanced polymorphism, as the protection provided by this G6PD deficiency against severe malaria in hemizygous males (and perhaps homozygous females) is also associated with risks of life-threatening complications, e.g., neonatal jaundice and devastating hemolytic crises precipitated by viral infections or ingestion of medicinal or dietary oxidants [23
]. In hemizygous males and homozygous females, the risk of hemolysis and protection against severe malaria would both reflect the presence of uniformly G6PD-deficient populations of erythrocytes, whereas in heterozygous females the relative reduction of these risks and lack of protection are attributable to mosaic populations of G6PD-normal and G6PD-deficient erythrocytes circulating in the bloodstream. Our evidence that the A− form of G6PD deficiency protects against severe malaria in its uniform (hemizygous, homozygous) but not mosaic (heterozygous) state appears more consistent than do previous proposals with currently hypothesized mechanisms of protection. Enhanced phagocytosis of parasitized erythrocytes [11
] or effects on the pathogenic consequences of parasitized erythrocytes in the microcirculation [24
] would be expected to operate preferentially in individuals whose erythrocytes are uniformly deficient in G6PD.