α Thalassemia

The first studies in the molecular era of the interplay between thalassemia and P falciparum malaria investigated this interaction in the Southwest Pacific. Previous demographic data had shown that malaria reaches its highest frequency in Papua New Guinea and then declines in a south-easterly direction. The frequency of α ⁺ thalassemia was found to also have a clinal distribution across this region, being highest in the coastal regions of Papua New Guinea, with a carrier rate of approximately 70%, and shifting to less than 5% in New Caledonia. Of course, this slowly declining frequency of α thalassemia mirroring a fall in the frequency of malaria transmission could be explained by the gene having been introduced from the Asian mainland and carried south by the early populations that spread across this region, hence being diluted as they moved further south. However, analysis of the molecular forms of α thalassemia and their particular HBA haplotypes provided clear evidence that the form of α thalassemia in this region was different from that of the Asian mainland and that it had almost certainly arisen locally and been amplified by a locally acting mechanism, presumably malaria. The same form of α thalassemia was found in some, but not all, of the Pacific islands in which malaria had never been recorded. However, it was always the same mutation that had been found in the Vanuatu Islands and hence seemed to have been distributed around the Pacific as its islands were populated.

The causal relationship of the high gene frequencies of α thalassemia to protection against P falciparum malaria was confirmed by case-control studies in Papua New Guinea, which provided clear evidence that both heterozygotes and homozygotes for α ⁺ thalassemia are protected against the more serious complications of P falciparum malaria infection. Later, similar studies performed in Africa confirmed that α thalassemia offers considerable protection against the complications of P falciparum infection and that protection against malaria-associated anemia is also mediated in mild P falciparum infections accompanied by inflammation. In addition, an unexpected finding was that α thalassemia seems to protect children against infections other than malaria, both in Papua New Guinea and Kenya.

Several mechanisms have been described that at least partly explain the protective effect of α thalassemia against malarial infection. Studies of a large cohort of children in Vanuatu, an island with an extremely high rate of transmission of both P falciparum and P vivax malaria, suggested that babies with uncomplicated malaria and enlargement of the spleen had a higher frequency of α ⁺ thalassemia than age-matched normal babies. It was suggested that the early susceptibility to P vivax , which may reflect the more rapidly turning over population of red cells in α ⁺ thalassemic infants, may be acting as a natural vaccine by inducing cross-species protection against later P falciparum infection. Of course, this protective mechanism would only be relevant to populations in which both varieties of parasite exist.

At the cellular level, no evidence shows a reduced rate of invasion or growth of P falciparum in the red cells of individuals with α ⁺ or α ⁰ thalassemia. It has been found, however, that these cells consistently bind more malaria-immune globulin than normal cells. Furthermore, α thalassemic red cells infected with parasites are more susceptible to phagocytosis in vitro, and are less able than normal red cells to form rosettes, an in vitro phenomena whereby uninfected red cells bind to infected cells. Experts have observed that complement receptor 1 (CR1) expression, which is essential for rosette formation, is reduced on α thalassemia red cells, offering a plausible explanation for reduced rosetting and, because the amount of rosetting strongly correlates with the severity of malaria infection, for a protective mechanism. Furthermore, infected α thalassemic red cells are less able to adhere to human umbilical endothelial cells and are less susceptible to phagocytosis. Because rosetting and cytoadherence are mechanisms that may be involved in the sequestration of infected red blood cells, these findings together suggest that abnormalities of the α thalassemic red cell membrane may be of fundamental importance in protecting against the severe complications of malaria.

It has also been suggested that the relatively high red cell counts in heterozygotes, or particularly homozygotes, for α ⁺ thalassemia may provide a further mechanism for protection, notably against the profound anemia that characterizes severe P falciparum infection in young infants.

The mechanism involved in protection against nonmalarial infections is unclear. It could be mediated through prevention of malaria-associated immune suppression or acquisition of nonspecific immunity against malaria that also protects against other diseases.

β Thalassemia

Apart from an early case-control study in northern Liberia, which suggested that the β thalassemia trait is protective against severe malaria, no further studies of this type have been performed. However, extremely suggestive evidence shows that the β thalassemias have reached their high frequency through selection against malaria. The comparative altitude studies mentioned earlier have been confirmed, showing a higher frequency of β thalassemia in the low-lying coastal regions of Papua New Guinea compared with frequencies in the mountainous regions.

In evolutionary terms, it seems likely that P f alciparum is a fairly recent human pathogen. A finding favoring β thalassemia being a relatively recently acquired polymorphism is the fact that every population in which this disease is common has a different set of mutations. Furthermore, studies of HBB haplotypes and their relationship to thalassemia mutations has provided further information in this respect. Unlike the HBA haplotypes, a “hot-spot” exists for recombination in the HBB cluster, resulting in distinct 5′ and 3′ haplotypes. Admixture between these haplotypes seems to have occurred among human populations, but this is not observed in the case of β thalassemia. The thalassemia mutations, which occur in the 3′ haplotype, are almost always associated with the same 5′ haplotype, indicating that they arose much more recently in evolutionary terms and that there has not been time for admixture of the haplotypes that carry these genes. This theory suggests a very recent selective pressure, approximately 5000 years, which agrees with current estimations of the time that human populations have been exposed to the pathogenic forms of Plasmodium .

In vitro culture studies have shown that the rates of invasion and growth of P falciparum in β thalassemic red cells do not differ significantly from those in normal cells. However, early studies showed that parasite growth is significantly retarded in red cells that contain more than 5pg/HbF per cell, an observation that was confirmed later using a transgenic mouse model carrying human γ genes. Because good evidence shows that the rate of decline of HbF production after birth is delayed in β thalassemia heterozygotes, this could provide a mechanism of protection during the first year of life.

Hemoglobin E

Because HbE is synthesized at a slightly reduced rate, homozygotes have the hematologic phenotype of the β thalassemia trait, whereas heterozygotes have very slightly reduced red cell indices. Because this variant reaches extremely high frequencies throughout parts of India and southeast Asia, reaching up to a 70% carrier frequency in parts of northern Thailand, it is highly likely that it has been influenced by strong selection, at least at some time during human evolution.

No formal case control studies have analyzed the putative protective effect of the HbE trait against malaria. However, extended linkage disequilibrium in the region of the HBB gene carrying the HbE variant provides strong evidence that this mutation has come under intense and relatively recent selective pressure. The HbE trait was found to be significantly associated with a reduced severity of disease in adults admitted with severe P falciparum malaria. In vitro culture evidence also shows that red cells from HbE heterozygotes, although not homozygotes, are more resistant to invasion by P falciparum .

Therefore, although the evidence is still incomplete, relative heterozygote resistance against severe malaria seems to be at least one of the important mechanisms responsible for the extraordinarily high gene frequencies of this variant in many Asian countries.

α Thalassemia

The first studies in the molecular era of the interplay between thalassemia and P falciparum malaria investigated this interaction in the Southwest Pacific. Previous demographic data had shown that malaria reaches its highest frequency in Papua New Guinea and then declines in a south-easterly direction. The frequency of α ⁺ thalassemia was found to also have a clinal distribution across this region, being highest in the coastal regions of Papua New Guinea, with a carrier rate of approximately 70%, and shifting to less than 5% in New Caledonia. Of course, this slowly declining frequency of α thalassemia mirroring a fall in the frequency of malaria transmission could be explained by the gene having been introduced from the Asian mainland and carried south by the early populations that spread across this region, hence being diluted as they moved further south. However, analysis of the molecular forms of α thalassemia and their particular HBA haplotypes provided clear evidence that the form of α thalassemia in this region was different from that of the Asian mainland and that it had almost certainly arisen locally and been amplified by a locally acting mechanism, presumably malaria. The same form of α thalassemia was found in some, but not all, of the Pacific islands in which malaria had never been recorded. However, it was always the same mutation that had been found in the Vanuatu Islands and hence seemed to have been distributed around the Pacific as its islands were populated.

The causal relationship of the high gene frequencies of α thalassemia to protection against P falciparum malaria was confirmed by case-control studies in Papua New Guinea, which provided clear evidence that both heterozygotes and homozygotes for α ⁺ thalassemia are protected against the more serious complications of P falciparum malaria infection. Later, similar studies performed in Africa confirmed that α thalassemia offers considerable protection against the complications of P falciparum infection and that protection against malaria-associated anemia is also mediated in mild P falciparum infections accompanied by inflammation. In addition, an unexpected finding was that α thalassemia seems to protect children against infections other than malaria, both in Papua New Guinea and Kenya.

Several mechanisms have been described that at least partly explain the protective effect of α thalassemia against malarial infection. Studies of a large cohort of children in Vanuatu, an island with an extremely high rate of transmission of both P falciparum and P vivax malaria, suggested that babies with uncomplicated malaria and enlargement of the spleen had a higher frequency of α ⁺ thalassemia than age-matched normal babies. It was suggested that the early susceptibility to P vivax , which may reflect the more rapidly turning over population of red cells in α ⁺ thalassemic infants, may be acting as a natural vaccine by inducing cross-species protection against later P falciparum infection. Of course, this protective mechanism would only be relevant to populations in which both varieties of parasite exist.

At the cellular level, no evidence shows a reduced rate of invasion or growth of P falciparum in the red cells of individuals with α ⁺ or α ⁰ thalassemia. It has been found, however, that these cells consistently bind more malaria-immune globulin than normal cells. Furthermore, α thalassemic red cells infected with parasites are more susceptible to phagocytosis in vitro, and are less able than normal red cells to form rosettes, an in vitro phenomena whereby uninfected red cells bind to infected cells. Experts have observed that complement receptor 1 (CR1) expression, which is essential for rosette formation, is reduced on α thalassemia red cells, offering a plausible explanation for reduced rosetting and, because the amount of rosetting strongly correlates with the severity of malaria infection, for a protective mechanism. Furthermore, infected α thalassemic red cells are less able to adhere to human umbilical endothelial cells and are less susceptible to phagocytosis. Because rosetting and cytoadherence are mechanisms that may be involved in the sequestration of infected red blood cells, these findings together suggest that abnormalities of the α thalassemic red cell membrane may be of fundamental importance in protecting against the severe complications of malaria.

It has also been suggested that the relatively high red cell counts in heterozygotes, or particularly homozygotes, for α ⁺ thalassemia may provide a further mechanism for protection, notably against the profound anemia that characterizes severe P falciparum infection in young infants.

The mechanism involved in protection against nonmalarial infections is unclear. It could be mediated through prevention of malaria-associated immune suppression or acquisition of nonspecific immunity against malaria that also protects against other diseases.

β Thalassemia

Apart from an early case-control study in northern Liberia, which suggested that the β thalassemia trait is protective against severe malaria, no further studies of this type have been performed. However, extremely suggestive evidence shows that the β thalassemias have reached their high frequency through selection against malaria. The comparative altitude studies mentioned earlier have been confirmed, showing a higher frequency of β thalassemia in the low-lying coastal regions of Papua New Guinea compared with frequencies in the mountainous regions.

In evolutionary terms, it seems likely that P f alciparum is a fairly recent human pathogen. A finding favoring β thalassemia being a relatively recently acquired polymorphism is the fact that every population in which this disease is common has a different set of mutations. Furthermore, studies of HBB haplotypes and their relationship to thalassemia mutations has provided further information in this respect. Unlike the HBA haplotypes, a “hot-spot” exists for recombination in the HBB cluster, resulting in distinct 5′ and 3′ haplotypes. Admixture between these haplotypes seems to have occurred among human populations, but this is not observed in the case of β thalassemia. The thalassemia mutations, which occur in the 3′ haplotype, are almost always associated with the same 5′ haplotype, indicating that they arose much more recently in evolutionary terms and that there has not been time for admixture of the haplotypes that carry these genes. This theory suggests a very recent selective pressure, approximately 5000 years, which agrees with current estimations of the time that human populations have been exposed to the pathogenic forms of Plasmodium .

In vitro culture studies have shown that the rates of invasion and growth of P falciparum in β thalassemic red cells do not differ significantly from those in normal cells. However, early studies showed that parasite growth is significantly retarded in red cells that contain more than 5pg/HbF per cell, an observation that was confirmed later using a transgenic mouse model carrying human γ genes. Because good evidence shows that the rate of decline of HbF production after birth is delayed in β thalassemia heterozygotes, this could provide a mechanism of protection during the first year of life.

Hemoglobin E

Because HbE is synthesized at a slightly reduced rate, homozygotes have the hematologic phenotype of the β thalassemia trait, whereas heterozygotes have very slightly reduced red cell indices. Because this variant reaches extremely high frequencies throughout parts of India and southeast Asia, reaching up to a 70% carrier frequency in parts of northern Thailand, it is highly likely that it has been influenced by strong selection, at least at some time during human evolution.

No formal case control studies have analyzed the putative protective effect of the HbE trait against malaria. However, extended linkage disequilibrium in the region of the HBB gene carrying the HbE variant provides strong evidence that this mutation has come under intense and relatively recent selective pressure. The HbE trait was found to be significantly associated with a reduced severity of disease in adults admitted with severe P falciparum malaria. In vitro culture evidence also shows that red cells from HbE heterozygotes, although not homozygotes, are more resistant to invasion by P falciparum .

Therefore, although the evidence is still incomplete, relative heterozygote resistance against severe malaria seems to be at least one of the important mechanisms responsible for the extraordinarily high gene frequencies of this variant in many Asian countries.