Article pp.55-64 du Vol.25 n°1-2 (2013)

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ARTICLE /ARTICLE

Red cell polymorphisms and malaria: an evolutionary approach*

Polymorphismes du globule rouge et paludisme : une approche évolutive

F. Bauduer

Revised 12 October 2011, Accepted 2 February 2012

Abstract Natural selection has made infectious agents a powerful driver of genome modification throughout human evolution. Malaria, a major killer in human socie- ties, is one of the best examples to illustrate this phenomenon. The parasite (Plasmodium) is transmitted to humans through the bites of mosquitoes of the genusAnophelesand develops its deleterious effects by infecting red blood cells (erythrocytes).Plasmodiumparasites date back to the dawn of mammalian evolution andP. falciparum, which causes the most severe form of malaria in humans, has a common evolutionary history with Homo sapiens. It has been suggested that malaria really became established with the change from hunter-gatherer to sedentary agricultural lifestyles about 10000 years ago. Micro- and macro- epidemiological surveys have shown that the geographical distributions offalciparummalaria and of some genetic red cell disorders such as thalassemias, hemoglobinopathies (Hb S, Hb C and Hb E), metabolic disorders (especially G6PD deficiency) and some abnormalities in membrane proteins largely overlap. These erythrocyte-associated mutations have increased in frequency because of the protection they offer against death from malaria, as demonstrated by in vitro models and/or clinical studies. They seem to be regionally specific and of recent origin, and are useful tools for population genetics, particularly for reconstructing the history of migrations. Furthermore, exposure to malaria has produced remarkable genetic variation which is by no means restricted to these red cell polymorphisms.

Keywords Malaria ·Plasmodium falciparum· Red blood cells · Natural selection

Résumé Par le biais de la sélection naturelle, les agents infectieux constituent une force essentielle agissant dans la modification du génome tout au long de l’évolution humaine. Le paludisme, cause majeure de mortalité pour l’humanité, représente l’un des meilleurs exemples illustrant ce phénomène. Le parasite (Plasmodium) est transmis aux êtres humains par la piqûre de moustiques du genreAnophe- leset développe son pouvoir pathogène au niveau des globules rouges. Les Plasmodiumsont apparus dès les débuts de la lignée des mammifères et l’espèce falciparum, agent causal de la forme la plus sévère de paludisme, partage une histoire évolutive commune avecHomo sapiens. Il est sup- posé que le paludisme s’est réellement installé lors des chan- gements d’habitud de vie correspondant au passage de la chasse et de la cueillette vers les pratiques agricoles et la sédentarisation il y a environ 10000 ans. Les études micro- et macroépidemiologiques ont montré l’existence d’un che- vauchement entre les distributions géographiques du paludisme àfalciparum et de certains désordres génétiques du globule rouge tels que les thalassémies, les hémoglobinopa- thies (Hb S, Hb C and Hb E), des perturbations métaboliques (en particulier le déficit en G6PD) ou des anomalies de pro- téines membranaires. Ces mutations associées au globule rouge ont gagné en fréquence du fait de l’avantage en survie en cas de paludisme comme démontré à la fois dans des modèles in vitroet/ou des études cliniques. Elles semblent être apparues récemment, avoir une spécificité géographique et constituent des outils précieux en génétique des populations pour reconstruire l’histoire des migrations. En outre, l’importante variabilité génétique reliée à l’exposition des populations au paludisme est loin d’être limitée aux polymorphismes du seul globule rouge.

Mots clésPaludisme ·Plasmodium falciparum· Globules rouges · Sélection naturelle

F. Bauduer (*)

Laboratoire de Génétique humaine

« Maladies rares, génétique et métabolisme », EA 4576, Université Bordeaux Segalen, Bordeaux & Service d’Hématologie,

Centre hospitalier de la Côte basque, Bayonne, France e-mail : [email protected]

*This work was presented in part at the 1836^thmeeting of the Société d’Anthropologie de Paris, Paris, January 26-28, 2011 DOI 10.1007/s13219-012-0060-8

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Many blood polymorphisms, especially the blood group sys- tems and HLA, have been studied in depth by population geneticists [1,2]. The distribution of these variants is a consequence of various factors: drift, migration and mixing, and natural selection [1]. Malaria, a major killer in human socie- ties due to a protozoa of the genusPlasmodiuminjected by a mosquito, is one of the best examples illustrating the selective power of infectious agents on the human genome [3].

Interestingly, it has been known for more than 60 years [4]

that the erythrocyte, the host cell playing a central role in the pathophysiology of human malaria, undergoes adaptive modifications in response to infection. Therefore, many of the protective mechanisms described so far involve genes affecting its structure or function. Abundant literature has been published on this topic in recent years, which will be reviewed in this article. Only the erythrocyte polymorphisms related to malaria resistance will be considered, as the evolution and selection of alleles in relation with other biological mechanisms is not within the scope of this paper.

Plasmodium species and malaria:

general characteristics

Overall, parasites of the genus Plasmodium include more than 200 species infecting reptiles, birds and mammals.

More than 25 distinct species have been described in pri- mates, which represent the majority of hosts among mammals. Human malaria is caused by five species: P. falciparum, P. vivax, P. ovale or P. malariae (these “classic” parasites, previously considered as“human-specific”, have also been found in great apes [5-7]) plusP. knowlesii(discovered more recently) [8,9]. The life cycle of these parasites begins with the injection of infectious forms (sporozoites) from theAnophelesmosquito into the blood of its host. Spor- ozoites enter liver cells and mature into schizonts. Each schizont generates a series of daughter forms (merozoites), which are able to invade naïve red cells. This penetration phase depends upon the interaction between ligands on the merozoite and receptors on the host cell membrane. Repeated phases of multiplication occur in erythrocytes. The asexual red cell stage is responsible for all of the symptoms and path- ological effects associated with malaria. Some merozoites may enter sexual stages during which they are transformed into gametocytes. The gametocyte is the infective form picked up by the mosquito. After fertilization, a zygote appears in the gut of theAnophelesthat subsequently develops into an oocyst outside the gut and finally generates a series of sporozoites in the salivary glands, which will be injected into a new host [10]. In malaria, the agglutination of parasite-infected red cells in the microvessels of the lung or the brain is the cause of lethal cases [11]. Currently, malaria causes an estimated 500 million clinical cases and almost one

million deaths (mainly during early childhood) per year throughout the tropical and subtropical zones [12,13]. This latter point is extremely relevant to natural selection. The malaria parasite and the red blood cell offer one of the best illustrations of the principles of evolution.

History of Plasmodium falciparum

The history of Plasmodium can be divided into two parts:

firstly, long-term co-evolution with its initial hosts and secondly, a relatively recent capture from apes at the time of human sedentarisation and population expansion. Both have shaped the human genome.

This protozoa has evolved in parallel with its successive hosts. P. falciparum, P. malariae, P. ovale and P. vivax descended from a single ancestor about 200 million years ago around the beginning of mammalian history [14].

P. malariae, P. ovale and P. vivax appeared between 76 and 160 million years ago and, 9 to 10 million years ago, P. reichenowi(infecting chimpanzees) diverged from P. falciparum around the time of separation between the human and great ape lineages.P. vivaxprobably originated in Asia and the falciparumspecies in Africa [14,15]. The origin and evolutionary history ofP. falciparumis still con- troversial and different hypotheses have been put forward.

According to Joy et al [16], the most recent common ancestor ofP. falciparummay be dated at around 100.000 years ago and a major expansion of this species occurred in the last 10.000 years. There are data to support this hypothesis.

According to the“out of Africa”paradigm, modern humans arose about 200.000 years ago in tropical Africa. They developed in association with the current genetic form of P. falciparum, which is thought to have originated in this geographical area, with subsequent distinct disseminations towards Southeast Asia and South America, according to investigations performed on mitochondrial DNA [17]. Anal- yses of intronic sequences revealing a low degree of variability suggest that P. falciparumemerged in its present form between 9.000 and 20.000 years ago [18]. This chronology is compatible with the idea that about 10.000 years ago (after the last glaciation), climate change associated with warm and humid conditions and the subsequent development of agriculture 5.000 to 10.000 years ago provided the right environmental conditions for the expansion of malaria.

Close cohabitation between humans and livestock favored the proliferation and diversification ofAnophelesmosquito vectors [19-22]. These relatively recent high levels of malaria endemicity in human history correlate well with the increase in population density that occurred with the adoption of agriculture and the distribution of current thalassemia molecular subtypes [23]. A so-called “co-speciation species”hypothesis has been proposed, including separate

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evolution ofP. reichenowiandP. falciparumin their hosts (hominins/chimps) that diverged from a common ancestor over the last 5-7 million years [24]. However, other recent phylogenetic investigations indicate that human P. falciparum strains seem to have evolved from a single cross- species transmission event. Rich et al [25] suggest thatP. fal- ciparumoriginated from the chimpanzee parasiteP. reich- enowiabout 3 million to 10.000 years ago. Analyses of mitochondrial sequences demonstrate thatP. falciparumis more probably of gorilla origin and not of chimpanzee, bonobo or ancient human origin [7]. The geographical dissemination of malaria occurred with human migrations, first throughout the Old World and then to the New World in association with explorations, commercial routes, missions and slavery.

In ancient times, malaria was known in China, India or Mesopotamia, as quoted in texts dating back more than three millennia. For instance, theNei Ching(Canon of Med- icine), a book commissioned by a Chinese Emperor about 4700 years ago, contains a description of malaria. Some data suggest that Alexander the Great could have died of this disease. In about 400 BC, Hippocrates described febrile disorders that are highly suggestive of various forms of plasmodial infections [21]. Malaria was markedly prevalent in Ancient Egypt as demonstrated by studies of mummies using different biological approaches [26]. Plasmodium spp. DNA was detected by PCR in 2 out of 91 Ancient Egyp- tian human remnants from necropolises dating back to the New Kingdom until the Late Period (1500-500 BC). The positive individuals also showed osteopathological evidence of chronic anemia [27]. The oldest genetic proof forfalci- parum malaria in precisely dated mummies was reported by Hawass et al [28] in a study on the 18^thRoyal Dynasty (circa 1550-1295 BC). Thus, plasmodial DNA was found in the famous Pharaoh Tutankhamun (circa 1333-1324 BC) and in his great-grandmother and great-grandfather, respectively named Thuya (circa 1410-1360 BC) and Yuya (circa 1410-1360 BC). Therefore,P. falciparum is likely to have had a significant impact on life expectancy in these times.

From the time of the Roman Empire to the end of the 19^th century, the history of malaria was influenced by a great many human migrations, especially the European conquest of the Americas and the slave trade. Before the advent of sanitary programs in the developed countries at the beginning of the 20^thcentury, malaria was more widely distributed (for instance in Europe where cases were found even in the northern regions) [29]. By this time, the parasitic condition was being eradicated in Europe and North America thanks to the discovery of the role of Anopheles mosquitoes in the transmission of the disease, the use of DDT insecticide, improvements in housing and living conditions and the availability of an efficient anti-malarial drug (quinine).

Unfortunately, mortality from malaria is still very high today in sub-Saharan Africa and South-East Asia [19,21,29].

Possible impacts of Plasmodium on human red cell polymorphisms: the 21^st century version of the “malaria hypothesis”

According to Kwiatkowski [30], P. falciparum is to be considered as“the strongest known force driving evolutionary selection in the recent history of the human genome” (Table 1). This parasite is able to kill before reproduction, especially children under the age of five. In the 1880s, Klebs and Tommassi-Crudeli noted in North America that individuals of African descent were more resistant to malaria than those from other origins [3]. Overall, selective pressure has led to the appearance of high frequencies of erythrocyte variants associated with lower malarial morbidity and mortality in exposed populations [31]. Up to now, a series of evolutionary anti-malarial“strategies”have been shown at the erythrocyte level:

•

inhibition of intracellular growth,

•

release of mature parasites or entry into the red cell,

•

promotion of immune response,

•

prevention of adherence of infected red cells with endothelium or other red cells (“rosetting”or“rosette formation”) within the microvessels (Fig. 1).

Haldane, one of the founders of population genetics, was the first to propose a relationship between a red cell polymorphism (thalassemia) and resistance to malaria. This became known as “the malaria hypothesis” [4]. Natural selection is the main cause of the extremely high frequency of these hemoglobin disorders [32]. It should be noted that inherited hemoglobin disorders are the most prevalent monogenic diseases in humans [23].

Quantitative hemoglobin disorders:

thalassemias

Thalassemias are characterized by the loss of either theαorβ chain of the globin, the proteic moiety of the hemoglobin molecule. Their geographical distribution is broad: around the Mediterranean Sea as initially noted by Haldane [4], Africa, the Southern part of Asia from the Middle East to China and the Western Pacific islands, which all represent present or past malarial zones [1,23,33].αthalassemia cases predominate in India/Oceania and β thalassemia is more prevalent in the Mediterranean basin and Asia. The underlying mutations are region-specific and have increased in frequency in the last 5000 years [23]. Thalassemias are associated with signs of chronic anemia on ancient bone specimens, such as porotic hyperostosis, which is a surro- gate indicator of selective malaria pressure for paleopatho- logists [20].αthalassemias result from impaired production of α globin. This protein is encoded by two (duplicated)

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genes. The α+ form retains one functional copy of these genes, both genes being inactivated in theα0 form. The clini- cally silent–α3.7 deletion is largely prevalent in sub-Saharan Africans whileα0-thalassemia is rare in this population [23].

Malaria protection afforded byαthalassemia has been more difficult to demonstrate than for Hb S, but a series of papers has suggested that this is indeed the case in African children for the more severe forms of the disease (especially in the case of severe anemia) [34-38].α+ thalassemias confer protection both in homo- and heterozygotes [34-36].αthalassemias are associated with a reduced expression of complement receptor 1 (CR1), a molecule that mediates the adhesion of parasitized red cells to uninfected ones [39]. A study in Papua New Guinea has shown that carriers also seem more resistant to upper respiratory tract infections during childhood [34]. Indi- viduals with homozygousα+ thalassemia also seem to be

protected against malaria because of a lower mean red cell volume, which reduces the overall parasite biomass [40]. α thalassemias are caused by many underlying mutations that are microgeographically specific, as shown in Papua New- Guinea (where they correlate with malaria according to alti- tude and latitude) or the Vanuatu Islands [23]. Theα3.7 III thalassemia can be used as a marker to reconstitute the peo- pling of Melanesia, Micronesia and Polynesia [23]. The Nepalese Tharu population has a lower prevalence of malaria than its neighbors, linked with a marked frequency ofαthal- assemia [41]. However, in some Pacific Islands, there are examples of high frequencies of α+ thalassemias in places where malaria has never been endemic [23]. Nevertheless, nearly all the cases ofα+ thalassemias seen in Polynesians can be accounted for by an initial mutation that was subsequently dispersed by migration [42].

Table 1 Red cell polymorphisms associated with genetic resistance to malaria (adapted from [21,23,30,102]) / Polymorphismes du globule rouge associés à une résistance au paludisme d’origine génétique (adapté de [21,23,30,102]).

Variants Geographic distribution Major protective phenotype

Structural hemoglobin variants

Hb S West Africa, Middle East,

parts of India

Reduced intra-erythrocyteP. falciparum development, increased clearance of infected red cells, better immune response againstP. falciparum

Hb C West Africa Reduced intra-erythrocyteP. falciparum

development and cytoadherence, increased immune clearance of infected red cells

Hb E East India, Southeast Asia Resistance to invasion byP. falciparum

Quantitative hemoglobin variants

Thalassemias Sub-Saharan Africa, Mediterranean zone,

Middle East, India, Southeast Asia

Reduced rosetting capacity

Membrane protein polymorphisms

Duffy blood group negativity Africa, Papua New Guinea Absence ofP. vivaxentry in red cell Gerbich group negativity (GP C null) Papua New Guinea Protection against the cerebral form

Blood group O Worldwide distribution with variable

frequencies

Reduced rosetting capacity

Band 3 gene deletions ovalocytosis Papua New Guinea Elective protection against cerebral form

CR1 polymorphisms Mali, Gambia, Thailand,

Papua New Guinea

Reduced cytoadherence ofP. falciparum infected red cells

Metabolic disorders

G6PD deficiency Tropical Africa, Middle East, tropical and subtropical Asia, Mediterranean, Papua New Guinea

Reduction of parasite replication

PK deficiency* Pre-modern malarial zones Resistance to red cell invasion

and increased clearance capacity of parasitized erythrocytes Abbreviations: see in text. *: variant of still uncertain significance.

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Theβglobin chain is encoded by a single gene.βthalas- semias are due to point mutations or small deletions or inser- tions (more than 200 have been found), which either abolish (β0) or only reduce (β+) the synthesis of functional protein.

Each population has its own spectrum of mutations. For instance, the IVS I: 110 (G→A) is mainly distributed in the Mediterranean zones and the IVS I: 5 (G→ C) in the Indian subcontinent [23]. Furthermore, a similar mutation seen in several unrelated populations is usually associated with different haplotypes, suggesting it corresponds to inde- pendent genetic events.βthalassemia may serve as an indicator of genetic isolation in some populations. For instance, almost all the cases found in the Jews of Kurdistan are related to only four mutations, three of which are unique to this ethnic group [43]. Likeα+ thalassemia,βthalassemia seems to offer some protection against malaria [23].

Structural hemoglobin disorders

These are due to point mutations inducing the modification of a single amino acid of the β globin chain: position 6 (Val→Glu) for Hb S, position 6 (Glu →Lys) for Hb C, position 26 (Glu→Lys) for Hb E.

Hemoglobin S (Hb S) or sickle cell trait

This allele is widely distributed throughout sub-Saharan Africa (especially in Cameroon, Guinea, Zaire, Uganda and Kenya), the Middle East (especially in the Qatif oasis

region in Saudi Arabia) and part of the Indian subcontinent, where the carrier frequency ranges from 5% to more than 40% of the population [23]. Some particular populations in Turkey (the Eti) or in Syria (the Khazramah) have high allele frequencies of 14% and 25% respectively [33]. Hb S is associated with an abnormal shape of the erythrocytes (sickle cells) with limited deformability and causing thromboses in the microvessels. Three African zones associated with founding mutations have been described on the West coast, in the centre West and centre South (the latter area being characterized by the Bantu language), with subsequent expansion occurring 2.000 to 2.500 years ago [44]. Accord- ing to Currat et al [45], haplotype studies show that the Hb S mutation from Senegal occurred between 45 to 70 genera- tions ago. Hb S cases found in Europeans (for instance in Portugal or Greece) or in Americans are due to recent migrations from Africa, especially during the slave trade [23].

In India, anthropological studies have suggested that Hb S arose from a single founding event in a population of the Indus Valley and was dispersed thereafter as a consequence of migrations during the last 5.000 years [46]. Hb S has never been described in Amerindians so far. Allison [47]

first raised the hypothesis that the sickle trait might be protective against malaria by conferring a selective advantage for heterozygotes. This was largely confirmed later [38,48,49] and Hb S now represents the paradigm of a so-called “balanced polymorphism”. Homozygosity for this allele causes a severe disorder that kills people before adulthood because of infection or thrombosis. Mechanisms for the Hb S protective effect are multiple and not fully understood. First, it has been shown that, in Hb S carriers, sickling rates are higher in parasitized erythrocytes than in non-parasitized cells. This phenomenon is thought to increase the clearance of infected red cells from the blood circulation [50]. Furthermore, when oxygen tension is low, P. falciparumparasites do not develop as well in Hb S carrying red cells as in the normal counterpart [51]. There is also an immune mechanism because Hb S carriers express higher titres of Ig G antibodies againstP. falciparumsurface antigens than normal individuals [52]. Overall, Hb S is considered as the most potent polymorphism relating to protection against severefalciparummalaria [38].

Hemoglobin C (Hb C)

Its distribution is restricted to West Africa especially and to a lesser extent to North Africa and the Mediterranean coasts.

The highest frequency of this variant is in the Ivory Coast [33].In vitro, erythrocytes carrying Hb C are associated with reduced development of the parasites [53] and demonstrate an impairment of adhesion to endothelial cells (the key event in severe malaria) because of abnormal surface expression ofPlasmodium falciparumErythrocyte Membrane Protein 1 Fig. 1 The four main evolutionary red cell-associated mechanisms

affording protection from Plasmodium in humans / Les quatre principaux mécanismes évolutifs de défense contre le genre Plasmodium impliquant le globule rouge chez l’homme

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(PfEMP-1) [54]. PfEMP1 encoded by thevargene family is the major parasite adhesin [55]. This molecule is highly immunogenic and following repeated exposures toP. falciparumin endemic areas, children acquire antibodies against the usual PfEMP1 variants that contribute to protection against severe disease [56]. Its erythrocyte ligand is CR1, which causes rosetting between infected and uninfected cells in the blood vessels. Clinical studies have shown that Hb C is protective against severe malaria [57-59]. It should be noted that, unlike the sickle cell mutation, homozygotes do not have significant clinical problems. Homozygosity offers better protection than heterozygosity [57-59]. Interest- ingly, the Dogon people from Mali are effectively protected against severe malarial attacks by Hb C although they have a low prevalence of Hb S [57].

Hemoglobin E (Hb E)

This variant is distributed across East India and Southeast Asia with a very high carrier frequency in North East Thailand (more than 70% among the So people), Laos and Cambodia (up to 50% among the Khmers) [23]. It represents the second most frequent hemoglobinopathy worldwide [33].

This polymorphism has been submitted to recent and intense selective pressure, since the most prevalent Asiatic variant found among the Thai reached its current frequency in less than 5000 years [60]. Heterozygotes are significantly less susceptible toPlasmodiuminvasionin vitro[61].

Metabolic disorders (enzymopathies)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency

This X-linked disorder is the most common human enzyme defect with about 400 million people carrying one of the causative mutations (more than 140 reported up to now).

Some G6PD deficient individuals develop acute hemolytic anemia following the ingestion of fava beans. This phenomenon has been noted since Antiquity and may explain the so-called“Pythagorean prohibition”on this bean [20]. The geographical distribution of G6PD deficiency (mainly tropical) parallels that of malaria, which is the main argument supporting the malaria protection hypothesis. Furthermore, a strong correlation between G6PD variants and distribution of the parasitic disease has been found on a small geographical scale in the Vanuatu archipelago [62]. The G6PD A- allele predominates in sub-Saharan Africa, where the G6PD Mediterranean variant is widely distributed. There is a clear reduction ofP. falciparum in vitro growth in these two types of deficient erythrocytes, as shown in the seminal work by Luzzatto et al [63] and subsequently confirmed by other investigators. Until the last decade, there were

conflicting findings about the level of protection conferred by G6PD A- hemizygosity in males and heterozygosity in females, respectively. Ruwende et al showed in 2002 that both conditions reduce the risk of severe malaria equally, by about 50% [64]. Overall, the level of protection seems comparable to that conferred by thalassemias. However, the biological bases of this protective effect are still not fully elucidated. It has been demonstrated by linkage analyses that the alleles conferring malarial resistance could have appeared recently, between 4.000 and 12.000 years ago [65].

This estimation correlates well with the time of emergence of P. falciparumas a major human pathogen proposed by Volkman et al [18]. P. falciparum and/or P. vivax could be the origin of G6PD-associated selection in African populations [21].

Pyruvate kinase (PK) deficiency

After G6PD deficiency, PK deficiency is the second most prevalent erythrocyte enzyme disorder worldwide (about one case per 20.000 individuals) and the leading cause of nonspherocytic hemolytic anemia. More than 150 mutations have been described in the corresponding PKLR gene.

Several arguments suggest that this enzymatic disease seems protective againstP. falciparum:

•

the distribution of PK deficiency cases parallels that of the pre-modern malarial area [29],

•

there are in vitro models in which homozygotes for the mutation are more resistant to red cell invasion and both homo- and heterozygotes demonstrate an increased capacity for clearing parasitized erythrocytes [66],

•

an in vivo model demonstrating resistance in PK deficient mice has been developed [67]. However, homozygosity is associated with severe clinical forms (profound transfusion-dependent anemia) and only heterozygosity could give a real selective advantage in malarial areas.

However, more data are needed to include PK deficiency as a candidate that fully matches the terms of the malaria hypothesis.

Membrane protein variants South Asian ovalocytosis

This phenotype characterized by an abnormal membrane shape is due to a band 3 gene mutation. This condition is mainly observed in Melanesians from Papua New Guinea (up to 10% of carriers). Homozygosity is lethal but selection favors the heterozygotes. Hence, although fully susceptible to infection, they are electively protected against the cerebral form, which is one of the main causes of death [68].

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Ovalocytic red cells infected by P. falciparum are more adhesive than normally shaped cells to the endothelial receptor CD36. This receptor is not expressed in the brain, which suggests that ovalocytosis may protect from cerebral malaria by distracting parasites from adhesion in the neurovascular system [69].

Duffy blood group negativity

The Duffy blood group gene shows a marked pattern of geographic differentiation between its three alleles FY*A, FY*B and FY*O (null allele) [70]. Duffy-negative erythrocytes (lacking a coreceptor forP. vivaxnamed DARC) resist invasion byP. vivaxmerozoites and confer protection to the host. This resistance trait is recessive and linked to a point mutation within the promoter of the DARC gene [71]. The FY*O allele is nearly fixed in sub-Saharan Africa [72] and absent in other parts of the world except in Papua New Guinea, as found more recently [73]. This allele has clearly been implicated as a target of natural selection [74]. Malaria related toP. vivaxis markedly less severe than that induced byP. falciparum. Why then is FY*O so prevalent in today African populations? This may be accounted for by more aggressiveP. vivax behavior in the past, a cross-protective effect againstP. falciparumor a selective advantage in an as yet unidentified agent. It should be noted that recent data indicate that someP. vivaxstrains may circumvent this adaptive phenomenon and are able to infect DARC negative erythrocytes [75,76]. Therefore, the absolute dependence on the presence of the Duffy group on the red cell forP. vivax infection and development no longer holds.

Blood group O

Several lines of evidence suggest that in the ABO system, group O has been directly influenced by selective pressure fromP. falciparumand offers an advantage in terms of morbidity and mortality in individuals infected by this parasite [77]. First, the group O allele evolved as a mutation from the group A gene before the migrations out of Africa, while P. falciparumwas prevalent in Hominids for a long time.

This point mutation (deletion of a guanosine at position 261) is linked to a founding effect and is common to all populations worldwide [78]. This indicates that the event took place before the earlyH. sapiensmigrated out of Africa (chronological argument). Secondly, there are arguments from clinical studies showing that patients with group O demonstrate milder manifestations and less severe outcomes than various non-O hosts [79-82]. Third, cytoadhesion of group O erythrocytes lacking both A and B surface antigens is largely reduced as shown by many teams using anin vitro rosette formation assay [11,83-85]. Interestingly, this phenomenon of cytoadhesion represents the key step in the

pathophysiology of lethal malaria associated with P. falciparumin microvessels.

Glycophorin polymorphisms

Different strains of P. falciparum may use various blood group proteins as receptors. Therefore, different adaptive pat- terns appear according to the geography. Glycophorins (GP) A, B, C or D are receptors for some parasite strains. Among 15 different African ethnic groups, Ko et al have shown particular molecular changes within the GP A and GP B genes determining MN and Ss blood types [86]. In some populations of tropical Africa, the hybrid polymorphism GP A - GP B, called the Dantu phenotype, has been demonstrated to be protective in vitro [87]. There is a high degree of polymorphism in GP B across endemic malarial zones, this protein acting as a receptor for the corresponding P. falciparum ligand EBL-1. The Efe Pygmies from the Ituri forest in the Democratic Republic of Congo have the highest gene frequency of GP B-null (i.e. S-s-U- blood group) in the world, at 59% [88]. A deletion within the GP C gene associated with negativity for the Gerbich blood group is seen at very high frequencies (up to 45%) in endemic malarial areas in Papua New Guinea. According to in vitro assays, this confers protection against a subgroup of parasites using a particular invasion pathway seen in this region especially [89]. How- ever, this variant does not seem to modify the prevalence or density of parasitemia in symptomless individuals [90]. This mutation is also able to cause ovalocytosis [90].

Complement receptor 1 (CR1)

The implication of complement receptor proteins, particularly CR1 (CD35), in the pathophysiology of malaria has been evaluated but the results are quite confusing in some populations. Some polymorphisms are overrepresented in endemic regions of Asia but not in Africa [91]. The blood group Knops mutation Sl (a-), a particular polymorphism of CR1, is highly overexpressed in Malians (65%) in compari- son with Afro-Americans (39%) or European-Americans (1%). These data may suggest that this mutation could offer a protective advantage against severe malaria [92].

Conclusions

There is a fascinating co-evolutionary process between humans and Plasmodium species. Our review focused on red cell polymorphisms as targets of natural selection regarding malaria. Numerous polymorphisms are involved, many of them still to be discovered. Populations living in malarial zones demonstrate a series of erythrocyte variants which may act in synergy to reduce the virulence ofPlasmodium

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in the human organism and are the consequence of various founding effects according to geography. On the other hand, although sickle cell trait andαthalassemia induce resistance individually against malaria, inheriting both conditions cancels out the benefit (“negative epistasis”). This is a possible explanation for their different geographical distributions [88,89]. However, why some variants have reached such high frequencies in some areas and not others is difficult to explain. Why is Hb S Africa-centered and Hb E an“Asiatic”trait? Besides natural selection, consanguinity, which is widely prevalent in many areas where inherited red cell disorders are common, further increases their frequency [32].

Of course, human adaptation to malaria is not restricted to erythrocytes. HLA alleles are a major example of genetic variability related to the selective pressure of infectious agents and are of great interest regarding malaria [95]. For instance, West African carriers of the class I B53 allele and the class II DRB1*1352 are significantly more resistant to cerebral malaria and severe anemia [48]. TNFα, a cytokine secreted by a category of leukocytes, plays a major role in the immune response. Some polymorphisms in the TNFα gene are clearly associated with an increased risk of severe clinical malaria [96,97]. As shown in this review, the adhesion of parasitized erythrocytes to the vascular endothelium is a phenomenon of paramount importance in the pathophysiology of severe malaria. Thus, genetic variation in some adhesion molecules such as CD36 [98] or intracellular adhesion molecule 1 (ICAM1) [99] interferes with susceptibility to the parasite. A polymorphism in the promoter region of the inducible nitric oxide synthase gene is associated with enhanced resistance to Plasmodium [100]. The ability to mount an antibody response is also a very important process as demonstrated in the Fulani people from West Africa [101].

Overall, these data are of interest not only for hematologists or biological anthropologists but also for medical professionals specializing in the prevention and treatment of this challeng- ing parasitic disease.

Acknowledgements :This work was supported by the association “Sang 64”. The author thanks the two anonymous reviewers for their careful examination of the paper. This paper is dedicated to Sophie, Andrea and Martha.

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