The mutation underlying the hemoglobin S variant (HbS) is a prototypical example of a balanced polymorphism: its frequency in populations is determined both by positive selection for heterozygosity (HbAS)5
and negative selection for homozygosity (HbSS), which causes sickle cell disease, a debilitating condition associated with chronic anemia and premature death. Negative selection has not been shown to affect the frequency of α+
-thalassemias. Perhaps as a consequence, these conditions approach fixation in a number of populations; for unknown reasons, however, frequencies remain relatively low in much of sub-Saharan Africa1
Despite conclusive evidence that both HbAS and α+
-thalassemia protect against severe and fatal P. falciparum
, the mechanisms underlying this protection are poorly understood. The mechanism of HbAS protection probably relates to either the physical or the biochemical properties of HbS-containing erythrocytes: in vitro
, invasion, growth and development of P. falciparum
parasites are all diminished in such cells at reduced oxygen tension6,7
, and parasite-infected HbAS erythrocytes also seem to be targeted for premature destruction by the spleen6,8,9
. These hypothetical mechanisms are supported by both the reduced incidence of clinical malaria and the lower parasite densities observed in children with HbAS erythrocytes when they suffer from clinical attacks3,10
. Much less is known about α+
-thalassemia. Although both heterozygosity (–α/αα) and homozygosity (–α/–α) with respect to the underlying mutation protect against severe and fatal malaria2,4
, neither protects against uncomplicated malaria11
or affects parasite densities during incident episodes2,12
. We believe that a better understanding of how these conditions protect against malaria might provide insights into both the pathophysiology of severe malaria and the host-parasite relationship more generally10
. Accordingly, we studied the effects of both HbAS and α+
-thalassemia on the epidemiology of malaria in children living on the coast of Kenya.
We measured the incidence of P. falciparum
malaria in two cohorts of children from Kilifi District, where almost 15% of the population has HbAS and the allele frequency of α+
-thalassemia is 0.50. In this area, malaria transmission occurs throughout the year, although most clinically evident infections present after the seasonal rains, which generally fall twice each year13
. Individually, both HbAS and α+
-thalassemia were associated with protection both from uncomplicated malaria, detected in the community by active clinical surveillance ( and ), and from more severe attacks resulting in hospital admission ( and ). For example, compared with a baseline group (children with both HbAA and normal thalassemia genotype, αα/αα), HbAS was >80% protective (incidence rate ratio (IRR) = 0.19; 95% confidence interval (c.i.) = 0.08–0.47; P
< 0.0001) and –α/–α was >30% protective (IRR = 0.67; 95% c.i. = 0.46–0.98; P
= 0.038) against hospital admission due to malaria ( and ). But we found evidence for a negative, epistatic interaction between the effects of HbAS and α+
-thalassemia on the incidence of malaria infection in both studies. In the case of each malaria outcome, the incidence was lowest in HbAS children with αα/αα; no additional advantage derived from coinherited –α/αα, and the protection afforded by HbAS was lost with coinherited –α/–α (– and ). Notably, of the 113 children in the birth cohort study with HbAS and αα/αα, none were admitted to hospital with severe malaria during almost 436 child years of follow-up (cyfu), whereas 5 of 46 HbAS and –α/–α children were admitted during only 183 cyfu (incidence = 27.33 episodes per 1,000 cyfu; P
= 0.0017 by Fisher’s exact test), an incidence similar to that observed in the baseline group. Given the small number of participants with these two-locus geno-types, this observation should be treated with caution, but it suggests that HbAS may be more protective than has been appreciated so far, failing to protect against malaria only when inherited with epistatic genes.
The incidence of uncomplicated malaria by hemoglobin type and α+-thalassemia genotype
IRRs for malaria by hemoglobin type and α+-thalassemia genotype. Derived from data described in and .
Hospital admissions with malaria and severe malaria by hemoglobin type and α+-thalassemia genotype
Parasite densities by hemoglobin type and α+-thalassemia genotype
Because previous work has shown that parasite densities are reduced by HbAS3,10
but not by α+
, we also analyzed parasitemia data. In keeping with our previous findings, we found that parasite densities were reduced during incident malaria infections in children with HbAS but not in children with α+
-thalassemia; however, in the case of all three clinical malaria outcomes, the density effect of HbAS was lost when coinherited with α+
-thalassemia (). Taken together, these observations are compatible with the conclusion that these two genes may be acting epistatically with regard to their malaria-protective effects. Individually, these observations might be considered somewhat weak, as several of the two-locus genotypes include relatively few individuals (reflected in the wide confidence intervals of our estimates for some of these groups). But we believe that our conclusion is strengthened both by the existence of a similar pattern in two separate studies, each involving different participants and measuring different outcomes, and by the independent observation of an effect at the level of parasite density.
The concept of epistasis holds that the fitness effect of an allele at one locus depends on the genotype coinherited at a second, unrelated locus. Although classic examples have been described in model systems14
, few descriptions are available in humans. This might be because both the outcome (e.g.
, for diseases such as myocardial infarction15
or inflammatory bowel disease18
) and the exposure (e.g.
, disease-susceptibility genes) are rare, making it difficult to test for epistasis without carrying out very large studies14
. The situation with malaria is different: several malaria-protective genes are found at high population frequencies, and the outcomes of interest, malaria or severe malaria, are relatively common. Therefore, malaria presents a unique opportunity to test for epistatic effects.
Epistasis is important for several reasons. On one hand, if it occurs commonly, epistatic interactions could make malaria-protective associations more difficult to detect and could complicate the search for mechanisms. On the other hand, epistatic interactions could provide a powerful tool for dissecting the mechanisms of protection afforded by known malaria-protective genes. For example, studying subjects with mixed genotypes for α+
-thalassemia and HbS could prove to be a valuable way to test specific hypotheses regarding the mechanisms of malaria protection in these conditions, such as reduced intracellular growth6,7
, reduced rosetting19
or increased opsonic phagocytosis8,9,20
. Our data, therefore, suggest a fresh approach to investigating malaria-protective mechanisms: studying the effects of genes when inherited in combination.
The frequencies of alleles underlying the hemoglobinopathies vary widely between malaria-endemic populations1
. For example, the α+
-thalassemias reach extreme frequencies in several populations in Oceania1,2
and South Asia21
, but despite levels of malaria transmission that are equally intense, they are much less common in sub-Saharan Africa12,22
. We asked whether the current balance of HbAS and α+
-thalassemia in malaria-endemic populations might be determined by epistasis. We addressed this question with a simple mathematical model, which we used to determine the allele frequencies at which HbS and α+
-thalassemia might equilibrate under different schedules of mortality, reflecting differing levels of negative epistasis. Under this model, increasing degrees of negative epistasis would affect equilibrium frequencies given scenarios of low () or high () ‘background’ rates of non-malaria-related mortality. Our simulations suggest that if epistasis were operating, it could result in a range of outcomes, including a state of equilibrium between α+
-thalassemia and HbAS at allele frequencies that depend on the selective forces involved. At low levels of epistasis, α+
-thalassemia could reach fixation; this situation would also be predicted to occur in the absence of HbS. Our simulations also suggest that if the selective advantage of HbAS alone were less pronounced (e.g.
, because the rate of non-malaria-related mortality was very high; ), negative epistasis could result in the extinction of HbAS. These results suggest, therefore, that epistasis might be one explanation for the wide variation in the frequency of these genes in malaria-endemic populations, a possibility that warrants further investigation.
Figure 2 The evolutionary consequences of negative epistasis between the malaria-protective effects of HbS and α+-thalassemia. The equilibrium frequencies of HbS (filled diamonds) and α+-thalassemia (unfilled circles) are shown under various schedules (more ...)
If this epistatic effect exists, the question of how it might be mediated remains. Several explanations seem plausible, two of which can be summarized as follows. First, the concentration of HbS is lower than that of HbA in erythrocytes from subjects with HbAS because α-globin has a greater affinity for normal (β) than for mutant (βs
) β-globin chains. With coexistent α+
-thalassemia, the relative deficiency of α-globin chains seems to magnify this affinity effect, such that the intraerythrocytic concentration of HbS is roughly halved in individuals with the two-locus genotype HbAS and –α/–α compared with those with HbAS and αα/αα23
. The malaria-protective effects of HbAS might include increased binding of hemichromes (degradation products of hemoglobin) to the cytoplasmic aspect of the erythrocyte membrane9
, leading to aggregation of the structural protein band 3. This, in turn, could result in opsonization by autologous IgG and complement C3c fragments and in the accelerated removal of infected erythrocytes by phagocytosis. This cascade could be critically dependent on the intracellular concentration of HbS; this possibility could easily be tested using cells from subjects with mixed genotypes. A second potential explanation relates to erythrocyte membrane physiology. The potential for P. falciparum
merozoite invasion might be critically dependent on the hydration status of target erythrocytes24
, a property determined by the physiological properties of their membrane. Although both α+
-thalassemia and HbS have predictable membrane effects individually, the effect of these conditions inherited in combination is less well understood and is an area of potential interest for future studies.
In summary, our data are compatible with the existence of an epistatic interaction between two common genetic conditions in humans, a situation that could explain, to some extent, the relative prevalence of HbAS and α+-thalassemia in malaria-endemic areas. Although such epistatic effects might impede the search for new malaria-protective associations, they might also provide needed insights into the mechanisms of action of malaria-protective genes.