For nearly a century after the publication of On the Origin of Species, there was not one example of natural selection that had been fully elucidated, that is, where the agent of natural selection was known, the effect on different genotypes could be measured, the genetic and molecular basis of variation was identified, and the physiological role of the gene or protein involved was well understood.

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The first such “integrated” example of natural selection on a molecular variant was elucidated in the 1950s, before the genetic code was even deciphered. Remarkably, this trailblazing work revealed natural selection operating on humans. It still stands today as one of the most detailed and important examples of evolution by natural selection in any species.

The story began when Tony Allison, a Kenyan-born Oxford medical student undertook a field of study of blood types among Kenyan tribes. One of the blood tests he ran was for sickle cells, red blood cells that form a sickle shape on exposure to the reducing agent sodium betasulfite or after standing for a few days (Figure 20-3). The deformed cells are a hallmark of sickle-cell anemia, a disease first described in 1910. These cells cause pathological complications by occluding blood vessels and lead to early mortality.

In 1949, the very year Allison went into the field, Linus Pauling’s research group demonstrated that patients with sickle-cell anemia had a hemoglobin protein with an abnormal charge (Hemoglobin S, or HbS) in their blood, compared with the hemoglobin of unaffected individuals (Hemoglobin A, or HbA). This was the first demonstration of a molecular abnormality linked to a complex disease. It was generally understood at the time that carriers of sickle cell were heterozygous and thus had a mixture of HbA and HbS (denoted AS), whereas affected individuals were homozygous for the Hb^S allele (denoted SS).

Allison collected blood specimens from members of the Kikuyo, Masai, Luo, and other tribes across the very diverse geography of Kenya. While he did not see any particularly striking association between ABO or MN blood types among the tribes, he measured remarkably different frequencies of H^S. In tribes living in arid central Kenya or in the highlands, the frequency of Hb^S was less than 1 percent; however, in tribes living on the coast or near Lake Victoria, the frequency of Hb^S often exceeded 10 percent and approached 40 percent in some locations (Table 20-1).

The allele frequencies were surprising for two reasons. First, since sickle-cell anemia was usually lethal, why were the frequencies of the Hb^S allele so high? And second, given the relatively short distances between regions, why was the Hb^S frequency high in some places and not others?

Allison’s familiarity with the terrain, tribes, and tropical diseases of Kenya led him to the crucial explanation. Allison realized that the Hb^S allele was at high frequency in low-lying humid regions with very high levels of malaria and nearly absent at high altitudes such as around Nairobi. Carried by mosquitoes, the intra-cellular parasite Plasmodium falciparum, which causes malaria, multiplies inside red blood cells (Figure 20-4). Mosquitoes and the disease are prevalent throughout sub-Saharan Africa in humid, low-lying regions near bodies of water where the mosquitoes reproduce. Allison surmised that the Hb^S allele might, by altering red blood cells, confer some degree of resistance to malarial infection.

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In order to test this idea, Allison carried out a much larger survey of Hb^S frequencies across eastern Africa, including Uganda, Tanzania, and Kenya. He examined about 5000 individuals representing more than 30 different tribes. Again, he found Hb^S frequencies of up to 40 percent in malarial areas and frequencies as low as 0 percent where malaria was absent.

The link suggested that the Hb^S allele might affect parasite levels, so Allison also undertook a study of the level of parasites in the blood of heterozygous AS children versus wild-type AA children. In a study of nearly 300 children, he found the incidence of malarial parasites was indeed lower in AS children (27.9 percent) than in AA children (45.7 percent) and that parasite density was also lower in AS children. The results indicated that AS children had a lower incidence and severity of malarial infection and would thus have a selective advantage in areas where malaria was prevalent.

The advantage to AS heterozygotes was especially striking in light of the disease suffered by SS homozygotes. Allison noted:

The proportion of individuals with sickle cells in any population, then, will be the result of a balance between two factors: the severity of malaria, which will tend to increase the frequency of the gene, and the rate of elimination of the sickle-cell genes in individuals dying of sickle-cell anaemia…. Genetically speaking, this is a balanced polymorphism [emphasis added], where the heterozygote has an advantage over either homozygote.⁵

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In other words, the sickle-cell mutation was under balancing selection (see Chapter 18) in areas where malaria was present. Positive selection operating on AS individuals is balanced by natural selection operating against AA individuals susceptible to malaria and SS individuals who would succumb to sickle-cell anemia.

How much of an advantage do AS individuals experience? This can be calculated by measuring the frequency of the Hb^S allele in populations and examining how these frequencies differ from the frequencies expected under the assumptions of the Hardy–Weinberg equation (see Chapter 18). A large-scale survey of 12,387 West Africans has revealed an Hb^S allele frequency (q) of 0.123. The frequencies calculated from the Hardy–Weinberg equation are lower for the homozygous phenotypes and higher for the heterozygous phenotype (Table 20-2). If it is assumed that the AS heterozygote has a fitness of 1.0, then the relative fitness of the other genotypes can be estimated from these differences. The relative fitness of the heterozygous AS genotype is 1.0/0.88 = 1.136, which corresponds to a selective advantage of approximately 14 percent.

This selective advantage has been well documented by long-term survival studies of AA, AS, and SS children in Kenya. These studies have found that AS individuals have a pronounced survival advantage over AA and SS individuals in the first few years of life (Figure 20-5).

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After Allison’s discovery, there was keen interest in determining the molecular basis of the difference(s) between Hb^S and Hb^A. Protein sequencing determined that Hb^S differs from Hb^A by just one amino acid, a valine in the place of a glutamic acid residue. This single amino acid change alters the charge of hemoglobins and causes it to aggregate into long rodlike structures within red blood cells. Once the genetic code was deciphered and methods for sequencing DNA were developed, Hb^S was determined to be caused by a single point mutation (CTC → CAC) in the glutamic acid codon encoding the sixth amino acid of the ß-globin subunit within the hemoglobin protein.

Interestingly, Allison also noted a high incidence of Hb^S outside of Africa, including in Italy, Greece, and India. Other blood-type markers did not indicate strong genetic relationships among these populations. Rather, Allison observed that these were also areas with a high incidence of malaria. The correlation between Hb^S frequency and the incidence of malaria held across not only East Africa, but the African continent, southern Europe, and the Indian subcontinent. Allison composed maps showing these striking correlations (Figure 20-6) and inferred that the Hb^S alleles in different regions arose independently, rather than through spreading by migration. Indeed, with the advent of tools for DNA geno-typing, it is clear that the Hb^S mutation has arisen independently in five different haplotypes and then increased to high frequency in particular regions. Based on the limited genetic diversity of malarial populations, it is believed that Hb^S mutations arose in just the past several thousand years, once populations began living around bodies of water with the advent of agriculture.

The role of Hb^S in conferring resistance to malaria illustrated three important facets of the evolutionary process: