Tnf

Figure 13.3 Genetic variation associated with malaria. Reprinted from Kwiatkowski (2005), copyright 2005, with permission from Elsevier.

a and ß thalassaemia

Malaria a and ß thalassaemia

Figure 13.4 Geographic relatedness of malaria and thalassaemia in Europe, Africa, and Asia. (A) The distribution of malaria (prior to major control programmes). (B) The distribution of a and p thalassaemia. Reprinted with permission from Weatherall (2008) .

Malaria

Figure 13.4 Geographic relatedness of malaria and thalassaemia in Europe, Africa, and Asia. (A) The distribution of malaria (prior to major control programmes). (B) The distribution of a and p thalassaemia. Reprinted with permission from Weatherall (2008) .

be reduced by about ten-fold (Modiano et al. 1991). The allele frequency was estimated at 0.8; it is suggested that it has not reached fixation only because of intermarriage with other ethnic groups (Modiano et al. 1991).

In the coastal region of northern Papua New Guinea the carrier frequency of a+ thalassemia varies between ethnic groups but in some groups was very high (Madang 0.68, Sepik 0.87) (Allen et al. 1997). A prospective case-control study in this region showed that a+ thalassemia in either the heterozygous or homozygous form was protective against severe malaria, as well as other infectious diseases (Allen et al. 1997). Odds ratios of 0.4 (0.22-0.74)

and 0.66 (0.37-1.2) were found for homozygotes and heterozygotes, respectively (Allen et al. 1997). Among African populations protection from severe malaria was also observed (Mockenhaupt et al. 2004; Williams et al. 2005e). In coastal Kenya, for example, a study of 655 cases of severe malaria and 648 controls showed protection associated with heterozygotes for a+ thalassemia with an odds ratio of 0.73 (0.57-0.94) and for homozygotes of 0.57 (0.40-0.81) versus controls; risk of death with severe malaria was also significantly reduced, by 40% and more than 60%, respectively (Williams et al. 2005e).

How a+ thalassemia acts to protect against severe malaria is unclear, it is not thought to involve effects on parasite growth or invasion but rather to involve differences in the red blood cell membrane and the immune response to infected cells (Weatherall 2008). It remains a complicated story. On the island of Espiritu Sano in Vanuatu in the southwest Pacific a paradox was seen with a higher incidence of uncomplicated malaria and prevalence of splenomegaly among children with a+ thalassemia, notably among younger children and those with P. vivax (Williams et al. 1996). Selection was postulated based on a possible cross species selective advantage in terms of immunity gained through infection with P. vivax providing protection for the more severe P. falciparum infection.

13.2.3 Malaria and structural haemoglobin variants

The single nucleotide substitution responsible for haemoglobin S (Hb S) is a further striking example of a balanced polymorphism in which a relatively high allele frequency is driven and maintained by the selective advantage conferred by possession of one copy of the variant allele (leading to sickle cell trait) in areas where human populations are exposed to the risk of malarial infection, counterbalanced by the deleterious effects of being homozygous for the Hb S allele (resulting in sickle cell disease). Hb S results from a single amino acid substitution, from glutamic acid to valine, due to an A to T transversion (rs334, c.20A>T) in the coding sequence of HBB in the p globin locus at chromosome 11p15.5 (Section 1.2). The variant is common in particular populations worldwide, notably in sub-Saharan Africa, the Middle East, and India with carrier frequencies of between 5% and 40% reported, and evidence that the mutation has occurred at least twice over our recent past (Pagnier et al. 1984; Weatherall 2008).

Early observations of a reduced prevalence of parasit-aemia on peripheral blood films and of enlarged spleens on clinical examination among individuals with sickle cell trait in Zimbabwe, together with reduced incidence of malaria among mine-workers with the trait, were suggestive of a protective role (Beet 1946; Brain 1952). In 1954, Allison reported that among Ugandan children the incidence of parasitaemia and parasite density was lower in those with sickle cell trait (Allison 1954b). Moreover, on a malaria parasite challenge study of adults who had been away from a malarial area for at least 18 months using two strains of P. falciparum, a striking difference was seen with 14 of 15 individuals without sickle trait becoming infected in contrast to only two of 15 with the trait, both with low parasitaemia (Allison 1954b). Allison also noted the geographic variation of the incidence of sickle cell trait within East Africa, being high in areas of hyperendemic malaria such as around Lake Victoria, in contrast to highland and other areas that were either malaria-free or subject to malaria epidemics where low frequencies were found (Allison 1954a).

Many studies have since shown association with protection from malaria for individuals with sickle cell trait (often denoted Hb AS) compared to individuals without a copy of the variant (Hb AA). For example Willcox and colleagues observed that the frequency of Hb AS was 1.8% among 558 patients attending hospital with malaria compared to 7.2% in the local population (P <0.001) (Willcox et al. 1983). Similar results were found in a large case-control study of 619 children with severe malaria in The Gambia by Hill and colleagues: the frequency of Hb S carriers was 1.2% compared to 12.9% among 510 mild malaria controls giving a relative risk of 0.08 (0.04-0.16), or more than 90% protection from severe malaria (Hill et al. 1991).

More recent studies from East Africa have also shown evidence of protection for individuals with sickle cell trait. A large birth cohort study in Kenya analysing survival between 2 and 16 months after birth showed reduced all-cause mortality associated with Hb AS compared to Hb AA with protection from severe malarial anaemia (Aidoo et al. 2002). Further insights were gained from the work of Williams and colleagues using large cohort studies of children in an area endemic for malaria on the coast of Kenya where 15% of the population has Hb AS. They demonstrated that the protective effect was specific to P falciparum infection rather than to other childhood diseases such as respiratory infections or gastroenteritis (Williams et al. 2005c). The data confirmed previous studies showing no association with symptomless parasitaemia but rather increasing degrees of protection dependent on the severity of illness: 50% protection for mild clinical malaria, 75% for admission with malaria to hospital, 86% for cerebral malaria, and nearly 90% for severe malarial anaemia. Parasite density was lower in cases of clinical malaria, four-fold lower in those with severe disease admitted to hospital for individuals Hb AS compared to Hb SS (Williams et al. 2005c). For mild clinical malaria, a clear age effect was seen with protection rising from 20% in the first 2 years of life, to 56% among children up to 10 years of age, then falling to 30% (Williams et al. 2005b).

Perhaps most intriguingly, the significant protective effects associated with Hb AS and with homozygosity for the a+ thalassaemia allele were lost when they were inherited together (Williams et al. 2005d). This appears to be an example of negative epistasis whereby the effect of an allele at one genomic locus depends on a genotype coinherited at a second unrelated locus. Such effects may be much more common than we appreciate. Why this should occur is unclear. It may be that the concentration of haemoglobin falls due to differences in binding affinity between the globin chains or that the deleterious effects on the parasite are lost in the presence of both polymorphisms. Looking for malaria susceptibility genes in a population becomes an even harder task as a consequence.

The functional basis for the observed relationship between Hb S and infection with P. falciparum has been the subject of much research. Parasitized red blood cells from individuals with sickle cell trait were shown to sickle much more readily than uninfected cells at varying oxygen concentrations, this is presumed to promote clearance of parasitized cells from the circulation by the spleen (Luzzatto et al. 1970; Roth et al. 1978). Parasite growth rates were reduced in low oxygen concentrations with intracellular death (Friedman 1978; Pasvol et al. 1978; Friedman et al. 1979). Mechanisms involving acquired immunity have been proposed with greater immune recognition of parasitized red blood cells among children with sickle cell trait (Marsh et al. 1989). Cabrera and colleagues, for example, found a highly significant association between Hb AS and immunoglobulin G (IgG) response to parasite variant surface antigens expressed on infected red blood cells (Cabrera et al. 2005).

Other structural variants of haemoglobin have been associated with malarial infection. Haemoglobin C arises due to a G to A single nucleotide substitution in HBB (rs33930165, c.19G>A) altering the same encoded amino acid as Hb S but rather than a glutamic acid to valine substitution there is a lysine amino acid substitution (Section 1.3.2). This variant is geographically concentrated among individuals living in central West Africa with, rarely, sporadic cases encountered elsewhere. Early work suggested a role for balancing selection by malaria (Allison 1956) but, in contrast to Hb S, there appear to be no negative consequences of possessing this variant such that it is considered as being subject to unidirectional positive selection in malarial regions. The polymorphism is currently found at relatively low allele frequencies, rarely greater than 20%, and is estimated to have arisen less than 5000 years ago (Wood et al. 2005). It may be that with time the allele frequency will rise to fixation and perhaps replace Hb S in particular populations subject to malarial selection (Hedrick 2004).

Studies of the Dogon people of Mali have shown that possessing one or two copies of Hb C is associated with protection from malaria with allele frequencies of 17.4% among uncomplicated malaria controls, 4.5% in severe malaria, and 2.9% among cerebral malaria cases (Agarwal et al. 2000). Moreover in a neighbouring West African country, Burkina Faso, a large case-control study of 4348 individuals showed a 29% reduction in risk of clinical malaria associated with heterozygosity, and 93% reduction with homozygosity, for Hb C (P = 0.008 and 0.001, respectively) (Modiano et al. 2001c). Reduced parasitaemia was noted with Hb C although effects on parasite growth remain controversial (Rihet et al. 2004; Williams 2006). A striking result was, however, reported by Fairhurst and colleagues who found reduced expression of the variant surface antigen Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) on the surface of infected red blood cells containing Hb C. Reduced adhesion to endothelium and other features were seen with Hb AC or Hb CC genotypes and it may be that the observed association of Hb C with severe malaria relates to reduced parasite sequestration and induction of inflammation in small blood vessels such as the brain

(Fairhurst et al. 2005). This process is thought to be critical to the development of cerebral malaria, a relatively rare but dangerous complication of P. falciparum infection in which patients rapidly enter a coma and often die.

13.2.4 Duffy antigen and vivax malaria

It is perhaps not surprising that genetic variation in the genes required for producing red blood cells and the haemoglobin they carry should have become so entwined with man's battle with malaria. For the malaria parasite, our red blood cells are an essential staging post in their life cycle as it is there that they differentiate into game-tocytes and are then taken up by a mosquito (Fig 13.2). Merozoites must gain entry into human red blood cells but the way may be shut. P. vivax parasites, for example, are unable to enter the red blood cells of people possessing a particular variant of a gene encoding a protein normally found on the red cell surface called the Duffy blood group antigen. Those individuals who lack the Duffy protein (Duffy blood group negative individuals) are completely protected from malaria due to this parasite. This explains the innate resistance of West Africans to P. vivax malaria (Miller et al. 1976). Given that the Duffy blood group is itself a benign genetic trait, it is believed that it was the selective pressure of malaria which drove the genetic variant conferring Duffy negative status to complete fixation in West African populations. It is notable however that malaria due to P. vivax is currently a much less severe disease than P. falciparum infection.

Duffy blood group antigen is a transmembrane glycoprotein encoded by the DARC gene (also known as the FY gene) on chromosome 1q21-q22. The protein also acts as a chemokine receptor for proinflammatory cytokines such as interleukin 18 (IL-18), hence the formal name of the gene product, 'Duffy blood group, chemokine receptor'. A single G to A nucleotide substitution in the coding sequence of the gene results in a structural change in the encoded protein from glycine to asparagine at amino acid position 44 (Tournamille et al. 1995b). The two alleles are denoted FY*A and FY*B. The Duffy negative pheno-type was only found with the FY*B allele among black Africans. Among individuals homozygous for the FY*B allele (denoted FY*Bnu" or FY*0), the DARC gene was not expressed. The effect is specific to red blood cells as the DARC gene continues to be expressed in other cell and tissue types. The molecular basis for this is very elegant and involves a further variant on the same haplotype as the coding variant defining FY*B (Fig. 13.6) (Tournamille et al. 1995a). The additional variant, an A to G single nucleotide substitution (rs2814778), occurs in the promoter region 46 bases from the transcriptional start site, and disrupts a consensus binding site for the transcription factor GATA-1. The change from AGATAA to AGGTAA dramatically reduces expression of the DARC gene. The beauty of this molecular process is that the specific transcription factor involved, GATA-1, is only found in red blood cells. In other cell types, the regulatory variant has no effect and therefore does not disrupt the cellular processes. It demonstrates how the consequences of genetic variation can be highly context-specific.

The FY*Bnull/FY*BnuN genotype, found in Duffy negative individuals, is at or near fixation in most West and Central African populations but is very rare outside Africa (Fig. 13.7). Given that vivax malaria is present at significant levels in non-African populations, it may be that in evolutionary terms the mutation occurred after the proposed major human migrations out of Africa (Box 8.5). Strong subsequent selection pressure could have resulted in non-African populations showing different and greater numbers of polymorphisms at the Duffy locus than in African populations. Greater genomic diversity among non-African populations is unusual, and contrasts with the majority of genomic regions where African populations are more genetically diverse (Hamblin and Di Rienzo 2000).

Remarkably, recent evidence suggests that exactly the same single nucleotide substitution which gives the Duffy negative phenotype has arisen independently in Papua New Guinea, only this time on the FY*A allele (Zimmerman et al. 1999). It has only been found in the heterozygous state (FY*A/FY*Anu"). When the red cells from these people are studied, they have on the cell surface only half the amount of Duffy antigen, consistent with a gene dosage effect: only one of the two alleles is prevented from expressing the gene due to loss of binding of the GATA-1 transcription factor. The genetic variant was found in the Abelam-speaking population of lowland Papua New Guinea exposed to all four malaria parasites. Only a small number of heterozygous individuals were found but the prevalence of vivax malaria was lower among FY*A/FY*Anu" genotypes compared to FY*A/FY*A.

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