Human genetic resistance to malaria

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[8] and ingest large amounts of hemoglobin.[9] 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.[10] This can result in changes in red cell membranes, including translocation of phosphatidylserine to their surface, followed by macrophage recognition and ingestion.[11] 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).[12] 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.

Sickle hemoglobin induces the expression of heme oxygenase-1 in hematopoietic cells. Carbon monoxide, a byproduct of heme catabolism by heme oxygenase-1(HO-1), prevents an accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of experimental cerebral malaria.[13] Other mechanisms, such as enhanced tolerance to disease mediated by HO-1 and reduced parasitic growth due to translocation of host micro-RNA into the parasite, have been described.[14]

Old World distribution of hemoglobin-inherited disorders

Malaria does not occur in the cooler, drier climates of the highlands in the tropical and subtropical regions of the world. 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. The frequencies of abnormal hemoglobins in different populations vary greatly, but some are undoubtedly polymorphic, having frequencies higher than expected by recurrent mutation. There is no longer doubt that malarial selection played a major role in the distribution of all these polymorphisms. All of these are in malarious areas,

• Sickle cell – The gene for HbS associated with sickle-cell is today distributed widely throughout sub-Saharan Africa, the Middle East, and parts of the Indian subcontinent, where carrier frequencies range from 5–40% or more of the population. Frequencies of sickle-cell heterozygotes were 20–40% in malarious areas of Kenya, Uganda, and Tanzania. Later studies by many investigators filled in the picture.[15][16] 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.

The thalassemias have a high incidence in a broad band extending from the Mediterranean basin and parts of Africa, throughout the Middle East, the Indian subcontinent, Southeast Asia, Melanesia, and into the Pacific Islands.

• α-thalassemia, which attains frequencies of 30% in parts of West Africa;[17]
• β-thalassemia, with frequencies up to 10% in parts of Italy;
• HbE, which attains frequencies up to 55% in Thailand and other Southeast Asian countries;[18] HbE is found in the eastern half of the Indian subcontinent and throughout Southeast Asia, where, in some areas, carrier rates may exceed 60% of the population.
• HbC, which attains frequencies approaching 20% in northern Ghana and Burkina-Faso. HbC is restricted to parts of West and North Africa.
• concurrent polymorphisms – 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.[19] 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.[19]

Sickle-cell disease was the genetic disorder to be linked to a mutation of a specific protein. Pauling introduced his fundamentally important concept of sickle cell anemia as a genetically transmitted molecular disease.[20]

This vein (4) shows the interaction between the malaria sporozoites (6) with sickle cells (3) and regular cells (1). While malaria is still affecting the regular cells (2), the ratio of sickle to regular cells is 50/50 due to sickle cell anemia being a heterozygous trait, so the malaria cannot affect enough cells with schizonts (5) to harm the body.

The molecular basis of sickle cell anemia was finally elucidated in 1959 when Ingram perfected the techniques of tryptic peptide fingerprinting. In the mid-1950s, one of the newest and most reliable ways of separating peptides and amino acids was by means of the enzyme trypsin, which split polypeptide chains by specifically degrading the chemical bonds formed by the carboxyl groups of two amino acids, lysine and arginine. Small differences in hemoglobin A and S will result in small changes in one or more of these peptides .[21] To try to detect these small differences, Ingram combined paper electrophoresis and the paper chromotography methods. By this combination he created a two-dimensional method that enabled him to comparatively "fingerprint" the hemoglobin S and A fragments he obtained from the tryspin digest. The fingerprints revealed approximately 30 peptide spots, there was one peptide spot clearly visible in the digest of haemoglobin S which was not obvious in the haemoglobin A fingerprint. The HbS gene defect is a mutation of a single nucleotide (A to T) of the β-globin gene replacing the amino acid glutamic acid with the less polar amino acid valine at the sixth position of the β chain.[22]

HbS has a lower negative charge at physiological pH than does normal adult hemoglobin. The consequences of the simple replacement of a charged amino acid with a hydrophobic, neutral amino acid are far-ranging, Recent studies in West Africa suggest that the greatest impact of Hb S seems to be to protect against either death or severe disease—that is, profound anemia or cerebral malaria—while having less effect on infection per se. Children who are heterozygous for the sickle cell gene have only one-tenth the risk of death from falciparum as do those who are homozygous for the normal hemoglobin gene. Binding of parasitized sickle erythrocytes to endothelial cells and blood monocytes is significantly reduced due to an altered display of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface.[23]

Protection also derives from the instability of sickle hemoglobin, which clusters the predominant integral red cell membrane protein (called band 3) and triggers accelerated removal by phagocytic cells. Natural antibodies recognize these clusters on senescent erythrocytes. Protection by HbAS involves the enhancement of not only innate but also of acquired immunity to the parasite.[24] Prematurely denatured sickle hemoglobin results in an upregulation of natural antibodies which control erythrocyte adhesion in both malaria and sickle cell disease.[25] Targeting the stimuli that lead to endothelial activation will constitute a promising therapeutic strategy to inhibit sickle red cell adhesion and vaso-occlusion.[26]

This has led to the hypothesis that while homozygotes for the sickle cell gene suffer from disease, heterozygotes might be protected against malaria.[27] Malaria remains a selective factor for the sickle cell trait.[28]

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 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 hemoglobin.[29]

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 hemoglobin 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.

The lower 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 resistance has been established to be homozygosity of α-Thalassemia gene within the local population.[30] Endogamy along caste and ethnic lines appear to have prevented these genes from being more widespread in neighboring populations.[31]

There is evidence that the persons with α-thalassemia, HbC and HbE have some degree of protection against the parasite.[17][32] Hemoglobin C (HbC) is an abnormal hemoglobin with substitution of a lysine residue for glutamic acid residue of the β-globin chain, at exactly the same β-6 position as the HbS mutation. The "C" designation for HbC is from the name of the city where it was discovered—Christchurch, New Zealand. People who have this disease, particularly children, may have episodes of abdominal and joint pain, an enlarged spleen, and mild jaundice, but they do not have severe crises, as occur in sickle cell disease. Haemoglobin C is common in malarious areas of West Africa, especially in Burkina Faso. In a large case–control study performed in Burkina Faso on 4,348 Mossi subjects, that HbC was associated with a 29% reduction in risk of clinical malaria in HbAC heterozygotes and of 93% in HbCC homozygotes. HbC represents a ‘slow but gratis’ genetic adaptation to malaria through a transient polymorphism, compared to the polycentric ‘quick but costly’ adaptation through balanced polymorphism of HbS.[33][34] HbC modifies the quantity and distribution of the variant antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the infected red blood cell surface and the modified display of malaria surface proteins reduces parasite adhesiveness (thereby avoiding clearance by the spleen) and can reduce the risk of severe disease.[35][36]

Hemoglobin E is due to a single point mutation in the gene for the beta chain with a glutamate-to-lysine substitution at position 26. It is one of the most prevalent hemoglobinopathies with 30 million people affected. Hemoglobin E is very common in parts of Southeast Asia. HbE erythrocytes have an unidentified membrane abnormality that renders the majority of the RBC population relatively resistant to invasion by P falciparum.[37]

Other genetic mutations besides hemoglobin abnormalities that confer resistance to Plasmodia infection involve alterations of the cellular surface antigenic proteins, cell membrane structural proteins, or enzymes involved in glycolysis.

Hemolytic anemia due to G6PD deficiency following Fava beans consumption

Glucose-6-phosphate dehydrogenase (G6PD) is an important enzyme in red cells, metabolizing glucose through the pentose phosphate pathway, an anabolic alternative to catabolic oxidation (glycolysis), while maintaining a reducing environment.[38] 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.[39] 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 second most common enzyme deficiency in humans (after ALDH2 deficiency), estimated to affect some 400 million people.[40] 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.[41] Mutant varieties of G6PD can be more unstable than the naturally occurring enzyme, so that their activity declines more rapidly as red cells age.

This question has been studied in isolated populations where antimalarial drugs were not used in Tanzania, East Africa[42] and in the Republic of the Gambia, West Africa, following children during the period when they are most susceptible to falciparum malaria.[43] 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.[44] An evolutionary genetic analysis of malarial selection of G6PD deficiency genes has been published by Tishkoff and Verelli.[41] The enzyme deficiency is common in many countries that are, or were formerly, malarious, but not elsewhere.

Pyruvate kinase (PK) deficiency, also called erythrocyte pyruvate kinase deficiency, is an inherited metabolic disorder of the enzyme pyruvate kinase. In this condition, a lack of pyruvate kinase slows down the process of glycolysis. This effect is especially devastating in cells that lack mitochondria because these cells must use anaerobic glycolysis as their sole source of energy because the TCA cycle is not available. One example is red blood cells, which in a state of pyruvate kinase deficiency rapidly become deficient in ATP and can undergo hemolysis. Therefore, pyruvate kinase deficiency can cause hemolytic anemia.

There is a significant correlation between severity of PK deficiency and extent of protection against malaria.[45]

Elliptocytosis, a blood disorder in which an abnormally large number of the patient's erythrocytes are elliptical. There is much genetic variability amongst those affected. There are three major forms of hereditary elliptocytosis: common hereditary elliptocytosis, spherocytic elliptocytosis and southeast Asian ovalocytosis.

stained red blood cell membrane proteins on electrophoresis gel strip[46]

Ovalocytosis is a subtype of elliptocytosis, and 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.[47] 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.[48] 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.

Red Blood Cell membrane major proteins

SAO is associated with protection against cerebral malaria in children because it reduces sequestration of erythrocytes parasitized by P. falciparum in the brain microvasculature.[49] 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.[50]

Plasmodium 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.[51] 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.[52] 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.[5] 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 Miller report needs qualification. In human studies of P. vivax transmission, there is evidence for the transmission of P. vivax among Duffy-negative populations in Western Kenya,[53] the Brazilian Amazon region,[54] and Madagascar.[55] The Malagasy people on Madagascar have an admixture of Duffy-positive and Duffy-negative people of diverse ethnic backgrounds.[56] 72% of the island population were found to be Duffy-negative. 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 indicated 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. More recently, Duffy negative individuals infected with two different 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. The frequency of such transmission is still unknown.[57] 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 same phenomenon has been observed in New World monkeys. [Note 1] However, DARC still appears to be a major receptor for human transmission of P. vivax.

The distribution of Duffy negativity in Africa does not correlate precisely with that of P. vivax transmission.[51] 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. 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.

Old World distribution of enzymopathies and immunogenetic variants

The Gerbich antigen system is an integral membrane protein of the erythrocyte and plays a functionally important role in maintaining erythrocyte shape. It also acts as the receptor for the P. falciparum erythrocyte binding protein. There are four alleles of the gene which encodes the antigen, Ge-1 to Ge-4. Three types of Ge antigen negativity are known: Ge-1,-2,-3, Ge-2,-3 and Ge-2,+3. persons with the relatively rare phenotype Ge-1,-2,-3, are less susceptible (~60% of the control rate) to invasion by P. falciparum. Such individuals have a subtype of a condition called hereditary elliptocytosis, characterized by oval or elliptical shape erythrocytes.

Rare mutations of glycophorin A and B proteins are also known to mediate resistance to P. falciparum.

Human leucocyte antigen (HLA) polymorphisms common in West Africans but rare in other racial groups are associated with protection from severe malaria. This group of genes encodes cell-surface antigen-presenting proteins and has many other functions. In West Africa, they account for as great a reduction in disease incidence as the sickle-cell hemoglobin variant. The studies suggest that the unusual polymorphism of major histocompatibility complex genes has evolved primarily through natural selection by infectious pathogens.

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.[60] However, HLA correlations vary, depending on the genetic constitution of the polymorphic malaria parasite, which differs in different geographic locations.[61][62]

Some studies suggest that high levels of fetal hemoglobin (HbF) confer some protection against falciparum malaria in adults with Hereditary persistence of fetal hemoglobin.[63]

The fitnesses of different genotypes in an African region where there is intense malarial selection were estimated by Anthony Allison in 1954.[69] 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. [Note 2] 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.[70] 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,[60] whereas in the Luo population of Kenya it was estimated that AS heterozygotes have 60% protection against severe malarial anemia.[67] 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.