Genetic resistance to malaria

Genetic resistance to malaria occurs through both modifications of the immune system that enhance immunity to this infection and also by changes in human red blood cells that hinder the malaria parasite's ability to invade and replicate within these cells. Host resistance to malaria therefore involves not only blood cell genes such as abnormal hemoglobins, Glucose-6-phosphate dehydrogenase deficiency, and Duffy antigens, which provide innate resistance, but also genes involved in immunity such as the major histocompatibility complex genes, which regulate adaptive immune responses. The resistance provided by modified blood cells aids survival through the dangerous years of early childhood, while the potent protection mediated by adaptive immune responses is more important in older children and adults living where malaria is endemic.

Malaria has placed the strongest known selective pressure on the human genome since the origination of agriculture within the past 10,000 years. Several inherited variants in erythrocytes have become common in formerly malarious parts of the world as a result of selection exerted by this parasite. This selection was historically important as the first documented example of disease as an agent of natural selection in humans. It was also the first example of genetically-controlled innate immunity that operates early in the course of infections, preceding adaptive immunity which exerts effects after several days. In malaria, as in other diseases, innate immunity leads into, and stimulates, adaptive immunity.

Population genetics
Evolution results from changes in gene frequencies in populations. The rates at which gene frequencies change, and the conditions under which they remain stable, were defined by the mathematical analyses of Ronald Fisher (The Genetical Theory of Natural Selection) and John Burdon Sanderson Haldane in the United Kingdom and Sewall Wright in the U.S.A. in the decade preceding the Second World War. Individuals of different genotypes vary in “fitness”, the probability that their genes will be passed on to the next generation. The term includes survival through reproductive age and relative fertility. Under some conditions genetic diversity (polymorphism) is stable, for example when a heterozygote has a fitness greater than that of either homozygote, whereas under other conditions polymorphism is unstable. Persons homozygous for abnormal hemoglobin (Hb) genes often have fitnesses lower than those with normal Hb, while heterozygotes have a greater fitness because of relative resistance to malaria, thereby maintaining stable polymorphisms in malarious environments.

Natural history of infections
In 2006 the World Health Organization estimated that there were about 250 million cases of malaria with 880,000 deaths. Approximately 90% of those who died were children in Africa infected with Plasmodium falciparum. Where this parasite is endemic young children have repeated malaria attacks. These are initially severe, and can be fatal, usually because of serious anemia or cerebral malaria. Repeated malaria infections strengthen adaptive immunity and broaden its effects against parasites expressing different surface antigens. By school age most children have developed efficacious adaptive immunity against malaria. These observations raise questions about mechanisms that favor the survival of most children in Africa while allowing some to develop potentially lethal infections. Evidence has accumulated that the first line of defense against malaria is provided by genetically-controlled innate resistance, mainly exerted by abnormal hemoglobins and glucose-6-phosphate dehydrogenase deficiency. In malaria, as in other infections, innate immune responses lead into, and stimulate, adaptive immune responses. However, the potent effect of genetically-controlled innate resistance is reflected in the probability of survival of young children in malarious environments. It is necessary to study innate immunity in the susceptible age group, younger than four years; in older children and adults the effects of innate immunity are overshadowed by those of adaptive immunity. It is also necessary to study populations in which random use of antimalarial drugs does not occur.

Sickle-cell
In 1910 a Chicago physician, James B. Herrick, observed sickle cells in the blood of an anemic dental student, Walter Clement Noel. In 1927 Vernon Hahn and Elizabeth Biermann Gillespie showed that sickling of the red cells was related to low oxygen. In some individuals this change occurs at partial pressures of Oxygen prevalent in the body, and produces anemia and other disorders, termed sickle-cell disease. In other persons sickling occurs only at very low partial pressures; these are asymptomatic sickle-cell trait carriers. The modern phase of research on this disorder was initiated by the famous chemist Linus Pauling in 1949. Pauling postulated that the hemoglobin (Hb) in sickle-cell disease is abnormal; when deoxygenated it polymerizes into long, thin, helical rods that distort the red cell into a sickle shape. In his laboratory electrophoretic studies showed that sickle-cell Hb (S) is indeed abnormal, having at physiological pH a lower negative charge than normal adult human Hb (A). In sickle-cell trait carriers there is a nearly equal amount of HbA and HbS, whereas in persons with sickle-cell disease nearly all the Hb is of the S type, apart from a small amount of fetal Hb. These observations showed that most patients with sickle-cell  disease are homozygous for the gene encoding HbS, while trait carriers are heterozygous for this gene. Persons inheriting a sickle-cell gene and another mutant at the same locus. e.g. a thalassemia gene, can also have a variant form of sickle-cell disease. Pauling also introduced the term “molecular disease”, which, together with “molecular medicine”, has become widely used. The next major advance was the discovery by Vernon Ingram in 1959 that HbS differs from HbA by only a single amino-acid substitution in the β-polypeptide chain (β6Glu → Val). It was later established that this results from a substitution of thymine for adenine in the DNA codon (GAG → GTG). This was the first example in any species of the effects of a mutation on a protein. Sickle Hb induces the expression of heme oxygenase-1 in hematopoietic cells. Carbon monoxide, a byproduct of heme catabolism by heme oxygenase-1, prevents an accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of experimental cerebral malaria.

Distribution of the sickle-cell gene
Since sickle-cell homozygotes are at a strong selective disadvantage, while protection against malaria favors the heterozygotes, it would be expected that high frequencies of the HbS gene would be found only in populations living in regions where malaria transmission is intense, or was so until the disease was eradicated. In a second study conducted in 1953 Allison showed that this was true in East Africa. Frequencies of sickle-cell heterozygotes were 20-40% in malarious areas, whereas they were very low or zero in the highlands of Kenya, Uganda, and Tanzania. Later studies by many investigators filled in the picture. High frequencies of the HbS gene are confined to a broad belt across Central Africa, but excluding most of Ethiopia and the East African highlands; this corresponds closely to areas of malaria transmission. Sickle-cell heterozygote frequencies up to 20% also occur in pockets of India and Greece that were formerly highly malarious. Tens of thousands of individuals have been studied, and high frequencies of abnormal hemoglobins have not been found in any population that was malaria free.

Independent origins of the sickle-cell gene
Two mutations found in nontranscribed sequences of DNA adjacent to the β-globin gene are so close to each other that the likelihood of crossover is very small. Restriction endonuclease digests of the β-globin gene cluster have shown five distinct patterns associated with the sickle-cell (GAG → GTG) mutation. Four are observed in Africa, the Bantu, Benin, Senegal and Cameroon types, and a fifth type is found in the Indian subcontinent and Arabia. The cited authors report that haplotype analysis in the β-globin region shows strong linkage disequilibrium over the distance indicated, which is evidence that the HbS mutation occurred independently at least five times. The high levels of AS in parts of Africa and India presumably resulted from independent selection occurring in different populations living in malarious environments. In summary, the demonstration that sickle-cell heterozygotes have some degree of protection against falciparum malaria was the first example of genetically- controlled innate resistance to human malaria, as recognized by experts on inherited factors affecting human infectious diseases. It was also the first demonstration of Darwinian selection in humans, as recognized by evolutionary biologists.

Other abnormal hemoglobins
The frequencies of abnormal hemoglobins in different populations vary greatly, but some are undoubtedly polymorphic, having frequencies higher than expected by recurrent mutation. Four of these are α-thalassemia, which attains frequencies of 30% in parts of West Africa; β-thalassemia, with frequencies up to 10% in parts of Italy; HbE (β26Glu → Lys), which attains frequencies up to 55% in Thailand and other Southeast Asian countries; and HbC (β6Glu → Lys), which attains frequencies approaching 20% in northern Ghana and Burkina-Faso. All of these are in malarious areas, and there is evidence that the persons with α-thalassemia, HbC and HbE have some degree of protection against the parasite. There is no longer doubt that malarial selection played a major role in the distribution of all these polymorphisms. An additional question is raised by the presence of polymorphisms for HbS and another Hb mutation in the sample population. Double heterozygotes for HbS and β-thalassemia, and for HbS and HbC, suffer from variant forms of sickle-cell disease, milder than SS but likely to reduce fitness before modern treatment was available. As predicted, these variant alleles tend to be mutually exclusive in populations. There is a negative correlation between frequencies of HbS and β-thalassemia in different parts of Greece and of HbS and HbC in West Africa. Where there is no adverse interaction of mutations, as in the case of abnormal hemoglobins and G6PD deficiency, a positive correlation of these variant alleles in populations would be expected and is found.

Thalassemias
It has long been known that a kind of anemia, termed thalassemia, has a high frequency in some Mediterranean populations, including Greeks and Southern Italians. The name is derived from the Greek words for sea (thalassa), meaning the Mediterranean sea, and blood (haima). Vernon Ingram again deserves the credit for explaining the genetic basis of different forms of thalassemia as an imbalance in the synthesis of the two polypeptide chains of Hb. In the common Mediterranean variant, mutations decrease production of the β-chain (β thalassemia). In α-thalassemia, which is relatively frequent in Africa and several other countries, production of the α-chain of Hb is impaired, and there is relative over-production of the β-chain. Individuals homozygous for β-thalassemia have severe anemia and are unlikely to survive and reproduce, so selection against the gene is strong. Those homozygous for α thalassemia also suffer from anemia and there is some degree of selection against the gene.

Glucose-6-phosphate dehydrogenase deficiency
Glucose-6-phosphate dehydrogenase (G6PD) is an important enzyme in red cells, metabolizing glucose through the pentose phosphate pathway and maintaining a reducing environment. G6PD is present in all human cells but is particularly important to red blood cells. Since mature red blood cells lack nuclei and cytoplasmic RNA, they cannot synthesize new enzyme molecules to replace genetically abnormal or ageing ones. All proteins, including enzymes, have to last for the entire lifetime of the red blood cell, which is normally 120 days. In 1956 Alving and colleagues showed that in some African Americans the antimalarial drug primaquine induces hemolytic anemia, and that those individuals have an inherited deficiency of G6PD in erythrocytes. G6PD deficiency is sex linked, and common in Mediterranean, African and other populations. In Mediterranean countries such individuals can develop a hemolytic diathesis (favism) after consuming fava beans. G6PD deficient persons are also sensitive to several drugs in addition to primaquine. G6PD deficiency is the most common enzyme deficiency in humans, estimated to affect some 400 million people. There are many mutations at this locus, two of which attain frequencies of 20% or greater in African and Mediterranean populations; these are termed the A- and Med mutations. Mutant varieties of G6PD can be more unstable than the naturally-occurring enzyme, so that their activity declines more rapidly as red cells age.

Malaria in G6PD-deficient subjects
This question has been studied in isolated populations where antimalarial drugs were not used in Tanzania, East Africa and The Gambia, West Africa, following children during the period when they are most susceptible to falciparum malaria. In both cases parasite counts were significantly lower in G6PD-deficient persons than in those with normal red cell enzymes. The association has also been studied in individuals, which is possible because the enzyme deficiency is sex-linked and female heterozygotes are mosaics due to lyonization, where random inactivation of an X-chromosome in certain cells creates a population of G6PD deficient red blood cells coexisting with normal red blood cells. Malaria parasites were significantly more often observed in normal red cells than in enzyme-deficient cells. An evolutionary genetic analysis of malarial selection on G6PD deficiency genes has been published by Tishkoff and Verelli. The enzyme deficiency is common in many countries that are, or were formerly, malarious, but not elsewhere.

South-East Asian ovalocytosis
Ovalocytosis is an inherited condition in which erythrocytes have an oval instead of a round shape. In most populations ovalocytosis is rare, but South-East Asian ovalocytosis (SAO) occurs in as many as 15% of the indigenous people of Malaysia and of Papua New Guinea. Several abnormalities of SAO erythrocytes have been reported, including increased red cell rigidity and reduced expression of some red cell antigens. SAO is caused by a mutation in the gene encoding the erythrocyte band 3 protein. There is a deletion of codons 400-408 in the gene, leading to a  deletion of 9 amino-acids at the boundary between the cytoplasmic and transmembrane domains of band 3 protein. Band 3 serves as the principal binding site for the membrane skeleton, a submembrane protein network composed of ankyrin, spectrin, actin, and band 4.1. Ovalocyte band 3 binds more tightly than normal band 3 to ankyrin, which connects the membrane skeleton to the band 3 anion transporter. These qualitative defects create a red blood cell membrane that is less tolerant of shear stress and more susceptible to permanent deformation. SAO is associated with protection against cerebral malaria in children because it reduces sequestration of erythrocytes parasitized by P. falciparum in the brain microvasculature. Adhesion of P. falciparum-infected red blood cells to CD36 is enhanced by the cerebral malaria-protective SAO trait. Higher efficiency of sequestration via CD36 in SAO individuals could determine a different organ distribution of sequestered infected red blood cells. These provide a possible explanation for the selective advantage conferred by SAO against cerebral malaria.

Resistance in South Asia
The lowest Himalayan Foothills and Inner Terai or Doon Valleys of Nepal and India are highly malarial due to a warm climate and marshes sustained during the dry season by groundwater percolating down from the higher hills. Malarial forests were intentionally maintained by the rulers of Nepal as a defensive measure. Humans attempting to live in this zone suffered much higher mortality than at higher elevations or below on the drier Gangetic Plain.

However, the Tharu people had lived in this zone long enough to evolve resistance via multiple genes. Medical studies among the Tharu and non-Tharu population of the Terai yielded the evidence that the prevalence of cases of residual malaria is nearly seven times lower among Tharus. The basis for their resistance to malaria is most likely a genetic factor. Endogamy along caste and ethnic lines appear to have confined these to the Tharu community. Otherwise these genes probably would have become nearly universal in South Asia and beyond because of their considerable survival value and the apparent lack of negative effects comparable to Sickle Cell Anemia.

Duffy antigen receptor
The malaria parasite Plasmodium vivax is estimated to infect 75 million people annually. P. vivax has a wide distribution in tropical countries, but is absent or rare in a large region in West and Central Africa, as recently confirmed by PCR species typing. This gap in distribution has been attributed to the lack of expression of the Duffy antigen receptor for chemokines (DARC) on the red cells of many sub-Saharan Africans. Duffy negative individuals are homozygous for a DARC allele, carrying a single nucleotide mutation (DARC 46 T → C), which impairs promoter activity by disrupting a binding site for the hGATA1 erythroid lineage transcription factor. In widely cited in vitro and in vivo studies, Miller et al. reported that the Duffy blood group is the receptor for P. vivax and that the absence of the Duffy blood group on red cells is the resistance factor to P. vivax in persons of African descent. This has become a well-known example of innate resistance to an infectious agent because of the absence of a receptor for the agent on target cells. However, observations have accumulated showing that the original report needs qualification. P. vivax can be transmitted in Squirrel monkeys (Saimiri boliviensis and S. sciureus), and Barnwell et al. have obtained evidence that P. vivax enters Saimiri monkey red cells independently of the Duffy blood group, showing that P. vivax has an alternative pathway for invading these cells. The Duffy binding protein, the one and only invasion ligand for DARC, does not bind to Saimiri erythrocytes although these cells express DARC and obviously become infected with P. vivax. The question is whether these observations are relevant to naturally occurring human transmission of P. vivax. Ryan et al. presented evidence for the transmission of P. vivax among a Duffy-negative population in Western Kenya. Independently, Cavasini et al. have reported P. vivax infections in Duffy antigen-negative individuals from the Brazilian Amazon region. P. vivax and Duffy antigen expression were identified by genotypic and other methods. A subsequent investigation in Madagascar has extended these observations. The Malagasy people in this island have an admixture of Duffy-positive and Duffy-negative people of diverse ethnic backgrounds. At eight sentinel sites covering different parts of the island 72% of the populations were Duffy-negative, as shown by genotyping and flow cytometry. P. vivax positivity was found in 8.8% of 476 asymptomatic Duffy-negative people, and clinical P. vivax malaria was found in 17 such persons. Genotyping of polymorphic and microsatellite markers suggested that multiple P. vivax strains were invading the red cells of Duffy-negative people. The authors suggest that among Malagasy populations there are enough Duffy-positive people to maintain mosquito transmission and liver infection. From this internal source P. vivax variants can develop, using receptors other than Duffy to enter red cells and multiply. More recently, Duffy negative individuals infected with two different strains (VK247 and classic strains) of P. vivax were found in Angola and Equatorial Guinea; further, P. vivax infections were found both in humans and mosquitoes, which means that active transmission is occurring. This finding reinforces the idea that this parasite is able to use receptors other than Duffy to invade erythrocytes, which may have an enormous impact in P. vivax current distribution. Because of these several reports from different parts of the world it is clear that some variants of P.vivax are being transmitted to humans who are not expressing DARC on their red cells. The frequency of such transmission is still unknown. Identification of the parasite and host molecules that allow Duffy-independent invasion of human erythrocytes is an important task for the future, because it may facilitate vaccine development.

P. vivax is clearly a less potent agent of natural selection that is P. falciparum. However, the morbidity of P. vivax is not negligible. For example, P. vivax infections induce a greater inflammatory response in the lungs than is observed in P. falciparum infections, and progressive alveolar capillary dysfunction is observed after the treatment of vivax malaria. Epidemiological studies in the Amazonian region of Brazil have shown that the number and rate of hospital admissions for P. vivax infections have recently increased while those of P. falciparum have decreased. Standard criteria for admission were used. The authors suggest that P. vivax infections in this region are becoming more severe. The distribution of Duffy negativity in Africa does not correlate precisely with that of P. vivax transmission. Frequencies of Duffy negativity are as high in East Africa (above 80%), where the parasite is transmitted, as they are in West Africa, where it is not. In summary, P. vivax can bind to and invade human and nonhuman primate erythrocytes through a receptor or receptors other than DARC. However, DARC still appears to be a major receptor for human transmission of P. vivax. The potency of P. vivax as an agent of natural selection is unknown, and may vary from location to location. DARC negativity remains a good example of innate resistance to an infection, but it produces a relative and not an absolute resistance to P. vivax transmission.

Agent of natural selection
Natural selection was traditionally attributed to phenomena such as competition for resources or predation. There was no example of natural selection operating on a common gene in humans, in contrast to selection against rare deleterious mutations. After the Second World War an Italian group (E.Silvestroni, I.Bianco and G.Montalenti) developed methods for identifying ß-thalassemia heterozygotes in populations, and recorded their frequencies in different parts of Italy. In some regions heterozygote frequencies up to 10% were observed, and the strong geographic correspondence between the incidence of thalassemia and endemic malaria was noted, as documented by an Italian historian of science. These researches “raised clearly the question of maintaining the frequency of a gene that, at that time, doomed homozygotes to death within the first two years of life”. At an international meeting in Italy in 1949 J.B.S.Haldane gave an address on “Disease and Evolution”. In the ensuing discussion Montalenti presented information on the distribution of thalassemia in Italy, and acknowledged a suggestion from J.B.S. Haldane that thalassemic heterozygotes may be resistant to malaria. Later in 1949 Haldane reiterated the same suggestion, with no reference to the Italian investigators. Haldane is therefore widely regarded as the originator of the “malaria hypothesis”. However, there have been suggestions that the role of Italian investigators in recognizing this correlation was insufficiently acknowledged, and that opinion was also expressed by the Nobel prizewinning geneticist Joshua Lederberg. Haldane’ general proposal that infections are important agents of natural selection was a timely reminder, but had a long parentage. It was first made by Alfred Russel Wallace, co-discoverer of natural selection as a cause of evolution, and in the first half of the twentieth century several examples of genes conferring resistance to infections, and their implications for natural selection, were published, as noted by Lederberg. Haldane conducted no research on abnormal hemoglobins or on malaria, and malaria was eradicated from Mediterranean countries after World War II, so the malaria hypothesis could not be validated on carriers of β-thalassemia.

Testing the malaria hypothesis
The first systematic investigation of the malaria hypothesis was conducted by Anthony Allison in East Africa in 1953. His initial study ascertained whether sickle-cell heterozygotes are protected against severe P.falciparum infections. This required working with children between four months and four years of age, when the morbidity and mortality from malaria is greatest. The study was done in Ugandan villages where antimalarial drugs were not used. Allison found that children in this age group carrying HbS had significantly lower malaria parasite counts than in those with HbA. Severe morbidity and mortality in malaria were known to be correlated with high parasite counts. This observation has been confirmed many times in different parts of Africa, and potentially lethal manifestations of malaria (cerebral malaria and severe anemia) are rare in sickle-cell heterozygotes. In the latter study the HbS carrier state was found to be negatively associated with all potentially lethal forms of P.falciparum malaria, whereas the negative associations of the carrier states of HbC and α-thalassemia were limited to cerebral malaria and severe anemia, respectively. These findings strongly suggest that, under conditions of intense P.falciparum transmission, young sickle-cell heterozygotes (AS) survive better than those with normal hemoglobin (AA), whereas sickle-cell homozygotes (SS) survive least well of all three genotypes. Detailed study of a cohort of 1022 Kenyan children living near Lake Victoria, published in 2002, confirmed this prediction. Many SS children still died before they attained one year of age. Between 2 and 16 months the mortality in AS children was found to be significantly lower than that in AA children. This well-controlled investigation shows the ongoing action of natural selection through disease in a human population. Analysis of genome-wide and fine-resolution association (GWA) is a powerful method for establishing the inheritance of resistance to infections and other diseases. Two independent preliminary analyses of GWA association with severe falciparum malaria in Africans have been carried out, one by the Malariagen Consortium in a Gambian population and the other by Rolf Horstmann (Bernhard Nocht Institute for Tropical Medicine, Hamburg) and his colleagues on a Ghanaian population. In both cases the only signal of association reaching genome-wide significance was with the HBB locus encoding the beta chain of hemoglobin, which is abnormal in HbS. This does not imply that HbS is the only gene conferring innate resistance to falciparum malaria; there could be many such genes exerting more modest effects that are challenging to detect by GWA because of the low levels of linkage disequilibrium in African populations. However the same GWA association in two populations is powerful evidence that the single gene conferring strongest innate resistance to falciparum malaria is that encoding HbS.

Mechanisms of protection
The mechanisms by which erythrocytes containing abnormal hemoglobins, or are G6PD deficient, are partially protected against P. falciparum infections are not fully understood, although there has been no shortage of suggestions. During the peripheral blood stage of replication malaria parasites have a high rate of oxygen consumption and ingest large amounts of hemoglobin. It is likely that HbS in endocytic vesicles is deoxygenated, polymerizes and is poorly digested. In red cells containing abnormal hemoglobins, or which are G6PD deficient, oxygen radicals are produced, and malaria parasites induce additional oxidative stress. This can result in changes in red cell membranes, including translocation of phosphatidylserine to their surface, followed by macrophage recognition and ingestion. The authors suggest that this mechanism is likely to occur earlier in abnormal than in normal red cells, thereby restricting multiplication in the former. In addition, binding of parasitized sickle cells to endothelial cells is significantly decreased because of an altered display of P.falciparum erythrocyte membrane protein-1 (PfMP-1). This protein is the parasite’s main cytoadherence ligand and virulence factor on the cell surface. During the late stages of parasite replication red cells are adherent to venous endothelium, and inhibiting this attachment could suppress replication.

Stimulation of adaptive immunity
One of the most interesting developments in biomedical science during the past few decades has been elucidation of mechanisms mediating innate immunity. One set of innate immune mechanisms is humoral, such as complement activation. Another set comprises pattern recognition receptors such as Toll-like receptors, which induce the production of interferons and other cytokines increasing resistance of cells such as  monocytes to infections. Cytokines produced during innate immune responses are among the activators of adaptive immune responses. Antibodies exert additive or synergistic effects with mechanisms of innate immunity. Unstable HbS clusters Band-3, a major integral red cell protein; antibodies recognize these clusters and accelerate their removal by phagocytic cells. Clustered Band 3 proteins with attached antibodies activate complement, and complement C3 fragments are opsonins recognized by the CR1 complement receptor on phagocytic cells. A population study has shown that the protective effect of the sickle-cell trait against falciparum malaria involves the augmentation of adaptive as well as innate immune responses to the malaria parasite, illustrating the expected transition from innate to adaptive immunity.

Fitnesses of different genotypes
The fitnesses of different genotypes in an African region where there is intense malarial selection were estimated by Anthony Allison in 1954. In the Baamba population living in the Semliki Forest region in Western Uganda the sickle-cell heterozygote (AS) frequency is 40%, which means that the frequency of the sickle-cell gene is 0.255 and 6.5 of children born are SS homozygotes. If the frequency of the heterozygote is 0.40 the sickle-cell gene frequency (q) can be calculated from the Hardy-Weinberg equation 2q(1-q) = 0,40, whence q = 0.2555 and q2, the frequency of sickle-cell homozygotes, is 0.065. It is a reasonable assumption that until modern treatment was available three quarters of the SS homozygotes failed to reproduce. To balance this loss of sickle-cell genes, a mutation rate of 1:10.2 per gene per generation would be necessary. This is about 1000 times greater than mutation rates measured in Drosophila and other organisms and much higher than recorded for the sickle-cell locus in Africans. To balance the polymorphism, Anthony Allison estimated that the fitness of the AS heterozygote would have to be 1.26 times than that of the normal homozygote. Later analyses of survival figures have given similar results, with some differences from site to site. In Gambians, it was estimated that AS heterozygotes have 90% protection against P.falciparum-associated severe anemia and cerebral malaria, whereas in the Luo population of Kenya it was estimated that AS heterozygotes have 60% protection against severe malarial anemia. These differences reflect the intensity of transmission of P.falciparum malaria from locality to locality and season to season, so fitness calculations will also vary. In many African populations the AS frequency is about 20%, and a fitness superiority over those with normal hemoglobin of the order of 10% is sufficient to produce a stable polymorphism.

Dating effects of malaria-related selection
Anthony Allison used the above fitness estimates to calculate the time that it would take for a newly mutated sickle-cell gene to increase in frequency in a population and attain a stable equilibrium. Under conditions of intense malarial selection the frequency of the sickle-cell gene would attain an equilibrium level in about 45 generations, slightly more than 1000 years. Under less intensive malarial selection the heterozygote advantage would be lower and a stable polymorphism with near 20% AS carriers (as commonly observed in Africa) would be attained in about 2,000 years. If malarial selection is relaxed, the frequency of the sickle-cell gene will fall exponentially. This has probably occurred in the African American population of the USA, but the rate of fall is uncertain because of the diverse and poorly documented African origin of the population as well as mixture in the USA with immigrants of other origins. Human genome sequencing can be applied not only to detect the effects of natural selection but also to obtain information about how recently it occurred. Sabeti and her colleagues have provided an appropriate framework. First, haplotypes at a locus of interest are identified (core haplotypes). Then the age of each core haplotype is assessed by the decay of its association to alleles at various distances from the locus, as measured by extended haplotype homozygosity (EHH). Core haplotypes that have unusually high EHH as well as a high population prevalence reveal the presence of mutations that rose to prominence in the gene pool faster than expected by random drift. When this approach was applied to the G6PD locus, significant evidence of selection was found. A linkage-disequilibrium test was used to estimate the date of origin of the mutated G6PD gene conferring resistance, which gave a figure of about 2,500 years. Haplotype diversity and linkage disequilibrium using microsatellite data had previously been applied by Tishkoff and her colleagues to estimate the dates of origin of G6PD variants. The African A- variant was estimated to have arisen within the past 3,840 to 11,760 years and the Med variant within the past 6,640 years. In summary, newly mutated abnormal hemoglobin and G6PD deficiency genes, arising in malarious environments, can quite rapidly become common and attain stable polymorphisms within 1,000 to 3,000 years, depending on the intensity of selection. Studies of genome variation and evolution of P.falciparum suggest that it originated within the last 3,200 to 7,700 years. These dates coincide with the spread of agriculture within the last 10,000 years, which increased the density of populations, forest clearing, and urbanization near sunlit pools of water. Such conditions favor the breeding of Anopheles mosquitos and the transmission of malaria. The dramatic changes in human social organization since the onset of the Neolithic Age have had equally striking effects on the transmission of infectious diseases, and these, in turn, have had selective effects on human genes and left their signatures on the human genome.

Genetic factors influencing adaptive immunity
Humoral and cell-mediated immune responses limit malaria parasite multiplication, and many cytokines contribute to the pathogenesis of malaria as well as to the resolution of infections. It is not surprising that genetic studies have identified several loci correlated with the severity of malaria. For example, polymorphisms at the HLA loci, which encode proteins that participate in antigen presentation, influence the course of malaria. In West Africa an HLA class I antigen (HLA Bw53) and an HLA class II haplotype (DRB1*13OZ-DQB1*0501) are independently associated with protection against severe malaria. However, HLA correlations vary, depending on the genetic constitution of the polymorphic malaria parasite, which differs in different geographic locations.

Some early contributions on innate resistance to infections of vertebrates, including humans, are summarized in Table 1.

'''Table 1. Examples of Genetically Controlled Innate Resistance to Infectious Agents or 	Recognition of their Products'''

It is remarkable that two of the pioneering studies were on malaria. Type 1 interferons and their mechanism of action have been analyzed in detail by genetic and other methods. The classical studies on the Toll receptor in Drosophila were rapidly extended to Toll like receptors in mammals and then to other pattern recognition receptors, which play important roles in innate immunity and its stimulation of adaptive immunity. The genetic control of innate and adaptive immunity is now a large and flourishing discipline. However, the early contributions on malaria remain as classical examples of innate resistance, which have stood the test of time.