Genetics, Vol. 162, 1849-1861, December 2002, Copyright © 2002

Nucleotide Variability at G6pd and the Signature of Malarial Selection in Humans

Matthew A. Saundersa, Michael F. Hammera, and Michael W. Nachmana
a Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721

Corresponding author: Matthew A. Saunders, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721., msaunder{at}u.arizona.edu (E-mail)

Communicating editor: R. G. HARRISON


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans. Deficiency alleles for this X-linked disorder are geographically correlated with historical patterns of malaria, and the most common deficiency allele in Africa (G6PD A-) has been shown to confer some resistance to malaria in both hemizygous males and heterozygous females. We studied DNA sequence variation in 5.1 kb of G6pd from 47 individuals representing a worldwide sample to examine the impact of selection on patterns of human nucleotide diversity and to infer the evolutionary history of the G6PD A- allele. We also sequenced 3.7 kb of a neighboring locus, L1cam, from the same set of individuals to study the effect of selection on patterns of linkage disequilibrium. Despite strong clinical evidence for malarial selection maintaining G6PD deficiency alleles in human populations, the overall level of nucleotide heterozygosity at G6pd is typical of other genes on the X chromosome. However, the signature of selection is evident in the absence of genetic variation among A- alleles from different parts of Africa and in the unusually high levels of linkage disequilibrium over a considerable distance of the X chromosome. In spite of a long-term association between Plasmodium falciparum and the ancestors of modern humans, patterns of nucleotide variability and linkage disequilibrium suggest that the A- allele arose in Africa only within the last 10,000 years and spread due to selection.

WITH the completion of the first drafts of the human genome (INTERNATIONAL HUMAN GENOME SEQUENCING CONSORTIUM 2001; VENTER et al. 2001 Down) considerable attention is now focused on understanding the levels and patterns of nucleotide variation among individuals. An accurate description of this variation is important for understanding processes of molecular evolution, for identifying disease genes, and for making inferences about the origin and history of Homo sapiens. A number of studies have described patterns of nucleotide variability in relatively large samples of individuals (HARDING et al. 1997 Down; CLARK et al. 1998 Down; DEINARD and KIDD 1998 Down; HARRIS and HEY 1999 Down, HARRIS and HEY 2001 Down; JARUZELSKA et al. 1999 Down; KAESSMANN et al. 1999 Down; RANA et al. 1999 Down; RIEDER et al. 1999 Down; FULLERTON et al. 2000 Down; GILAD et al. 2000 Down; HAMBLIN and DI RIENZO 2000 Down; NACHMAN and CROWELL 2000 Down; ZHAO et al. 2000 Down; ALONSO and ARMOUR 2001 Down; YU et al. 2001 Down), and a large public effort recently identified and mapped >1 million single-nucleotide polymorphisms (SNPs; INTERNATIONAL SNP MAP WORKING GROUP 2001). These studies have generally focused on regions of the genome in which positive natural selection is believed to be a negligible force, and as such, provide a baseline for average patterns of genomic variability. However, selection may have been an important force in shaping human genetic variation. Selection can have a powerful effect on patterns of linkage disequilibrium (LD), levels of heterozygosity, and frequencies of alleles segregating in a population, and these effects may extend to linked sites at considerable distances from the targets of selection (HUDSON 1990 Down, HUDSON 1996 Down). One way to study the impact of selection in shaping nucleotide variability is to look at regions of the genome in which the strength and form of selection are known and in which the connections from genotype to phenotype to environment are well understood.

The X-linked gene coding for glucose-6-phosphate dehydrogenase (G6PD) is subject to malarial selection in some human populations. The normal G6PD enzyme catalyzes a critical step in the pentose monophosphate shunt of glycolysis, and in cases of dysfunctional G6PD, an individual may suffer with clinical manifestations that include hemolytic anemia and neonatal jaundice (BEUTLER 1994 Down). Some human populations exhibit G6PD deficiency alleles at frequencies that range up to 65% (LIVINGSTONE 1985 Down; OPPENHEIM et al. 1993 Down). In general, there is a geographic correlation between the frequency of G6PD deficiency alleles and the historical prevalence of malaria globally (ALLISON 1960 Down; MOTULSKY 1961 Down; OPPENHEIM et al. 1993 Down). Moreover, in vitro studies (ROTH et al. 1983 Down; ROTH and SCHULMAN 1988 Down) and epidemiological evidence (RUWENDE et al. 1995 Down) indicate that G6PD deficiency confers some resistance to Plasmodium falciparum, the primary human malaria parasite.

The most common G6PD deficiency allele in sub-Saharan Africa is G6PD A-, and it typically reaches frequencies near 20% in populations living in malarial areas (LIVINGSTONE 1985 Down). The A- allele differs from the normal allele (G6PD B) by nonsynonymous changes at coding nucleotide positions 202 and 376. A minor deficiency allele, G6PD A+, differs from the B allele only at site 376 (Fig 1). The enzymatic activities of the A+ and A- alleles are 85 and 12% of normal levels, respectively (HIRONO and BEUTLER 1988 Down; BEUTLER et al. 1989 Down; VULLIAMY et al. 1991 Down). The mild deficiency phenotype characteristic of G6PD A+ does not cause significant clinical manifestations and does not appear to confer resistance to malaria (RUWENDE et al. 1995 Down). However, the deficiency phenotype characteristic of G6PD A- confers an ~50% reduction in risk of severe malaria in both heterozygote females and hemizygote males. Homozygous females probably have a similar level of protection from malaria, although this genotype is quite rare (RUWENDE et al. 1995 Down). In the presence of falciparum malaria, the G6PD A- allele is therefore beneficial, while in the absence of malaria this allele is deleterious. Thus G6pd provides a rare example of a gene in humans where the selective agent and approximate form and strength of selection are known (RUWENDE et al. 1995 Down; TISHKOFF et al. 2001 Down).



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  		Figure 1. Schematic ideogram of the human X chromosome and the genomic regions sampled in this study. Genes located between L1cam and G6pd on Xq28 are marked. Solid arrows indicate positions of the amplification primers Gf, Gr, Lf, and Lr. For G6pd, the mutations that define G6PD A+ and G6PD A- are marked at coding positions 376 and 202. L1cam is located 556 kb from G6pd. Exons are marked with solid boxes. Polymorphic amino acid residues for alleles G6PD B, G6PD A+, and G6PD A- due to nucleotide polymorphisms at sites 202 and 376 are shown in the shaded box.

As part of an ongoing project to characterize patterns of nucleotide variability at multiple loci throughout the genome for a common worldwide sample of human DNAs and to investigate the impact of selection on G6pd, we sequenced 5.1 kb of G6pd in a sample of 47 humans (Table 1). We also sequenced 3.7 kb at L1cam in these same individuals. L1cam is situated 556 kb from G6pd; thus, polymorphisms at L1cam provide an opportunity to investigate the impact of selection on neighboring sites. Our nucleotide data suggest that the effects of selection on G6pd are more subtle than those predicted under a model of long-term diversifying selection.


 
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  		Table 1. Polymorphisms for a worldwide sample of humans at G6pd and L1cam


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Samples:
DNA sequences were determined in a sample of 41 human males, including 10 from Africa, 10 from the Americas, 10 from Europe, and 11 from Asia and Melanesia (Table 1). This sample was chosen as part of a long-term project in our labs to survey nucleotide variability at a number of loci throughout the genome using a common set of individuals (e.g., NACHMAN et al. 1998 Down; NACHMAN and CROWELL 2000 Down; M. W. NACHMAN and M. F. HAMMER, unpublished data). However, since G6PD A- alleles are primarily found in Africa and since the effects of selection at G6pd are likely to be found primarily in Africa, we augmented our worldwide sample with 4 additional African individuals that were known [by restriction fragment length polymorphism (RFLP) analysis] to carry G6PD A- alleles and 2 individuals known to carry G6PD A+ alleles. This allowed us to investigate patterns of variability within G6PD A- alleles and to study LD between G6PD A- alleles and other alleles. Homologous sequences from a male chimpanzee (Pan troglodytes) and a male orangutan (Pongo pygmaeus) were also determined for divergence estimates. By studying X-linked loci in males we were able to PCR amplify single alleles and to directly recover haplotypes over long genomic distances to study patterns of linkage disequilibrium.

PCR amplification and sequencing:
Maps of the human X chromosome and the loci sampled in this study, G6pd and L1cam, are presented in Fig 1. L1cam was chosen because of its proximity to G6pd (556 kb); all polymorphisms detected at L1cam are silent or noncoding, and there is no a priori reason to assume that L1cam itself is a target of selection. Approximately 82 other genes are found within 1 Mb on either side of G6pd and none of these genes are known to be recent targets of positive selection. PCR fragments were amplified for G6pd (5.2 kb) and L1cam (4.2 kb) using a long-template PCR system (Roche Biochemicals). For G6pd, the primers Gf (5' GTT TAT GTC TTC TGG GTC AGG GAT GG 3') and Gr (5' AGT GTT GCT GGA AGT CAT CTT GGG T 3') are positioned with the 5' end of the primer at sites 206322 and 201052, respectively, in GenBank accession no. L44140. For L1cam, the primers Lf (5' TCC TCT CCA GAG TAG CCG ATA GTG ACC 3') and Lr (5' AAG TTT CTA CTG GCC TGA CCC TCT CG 3') are positioned with the 5' end of the primer at sites 19587 and 24251, respectively, in GenBank accession no. U52112 (Fig 1). Internal primers (available upon request) were used to generate overlapping sequence runs on an ABI 377 automated sequencer. A contiguous sequence that included coding and noncoding regions (5109 and 3691 bp for G6pd and L1cam, respectively) was assembled for each individual and aligned using the computer program Sequencher (Gene Codes, Ann Arbor, MI). Sequences have been submitted to GenBank under accession nos. AY158094, AY158095, AY158096, AY158097, AY158098, AY158099, AY158100, AY158101, AY158102, AY158103, AY158104, AY158105, AY158106, AY158107, AY158108, AY158109, AY158110, AY158111, AY158112, AY158113, AY158114, AY158115, AY158116, AY158117, AY158118, AY158119, AY158120, AY158121, AY158122, AY158123, AY158124, AY158125, AY158126, AY158127, AY158128, AY158129, AY158130, AY158131, AY158132, AY158133, AY158134, AY158135, AY158136, AY158137, AY158138, AY158139, AY158140, AY158141, AY158142 and AY167680, AY167681, AY167682, AY167683, AY167684, AY167685, AY167686, AY167687, AY167688, AY167689, AY167690, AY167691, AY167692, AY167693, AY167694, AY167695, AY167696, AY167697, AY167698, AY167699, AY167700, AY167701, AY167702, AY167703, AY167704, AY167705, AY167706, AY167707, AY167708, AY167709, AY167710, AY167711, AY167712, AY167713, AY167714, AY167715, AY167716, AY167717, AY167718, AY167719, AY167720, AY167721, AY167722, AY167723, AY167724, AY167725, AY167726, AY167727, AY167728 for G6pd and L1cam, respectively.

Data analysis:
Nucleotide diversity, {pi} (NEI and LI 1979 Down), and the proportion of segregating sites, {Theta} (WATTERSON 1975 Down), were calculated using the program PROSEQ (FILATOV et al. 2000 Down) for the worldwide sample, for African individuals, and for non-African individuals. Only the 41 individuals of the nonaugmented worldwide sample were included in analyses of nucleotide diversity, and insertion-deletion polymorphisms were excluded. Under neutral equilibrium conditions both {pi} and {Theta} estimate the neutral parameter 3Neµ for X-linked loci, where Ne is the effective population size and µ is the neutral mutation rate. Tajima's D (TAJIMA 1989 Down), Fu and Li's D (FU and LI 1993 Down), and Fay and Wu's H (FAY and WU 2000 Down; http://crimp.lbl.gov/htest.html) were calculated to test for deviations from a neutral equilibrium frequency distribution for both loci. Ratios of polymorphism to divergence for G6pd and L1cam were compared with the expectations under a neutral model using the Hudson-Kreitman-Aguadé (HKA) test (HUDSON et al. 1987 Down). Polymorphism data for these tests were derived from the 41 sequences determined in this study for G6pd and L1cam, as well as from Dmd (intron 44) from the same set of individuals (NACHMAN and CROWELL 2000 Down) and from the Pdha1 data of HARRIS and HEY 1999 Down. Dmd and Pdha1 were chosen for comparison because they both reside in regions of the X chromosome with moderate to high rates of recombination and thus are expected to be relatively free of the effects of selection at linked sites. Divergence data were derived for each of these loci by comparing the homologous sequences from a chimpanzee to a single randomly chosen human allele. LD between pairs of polymorphic sites was measured using the statistics D' (LEWONTIN 1964 Down) and r2 (HILL and ROBERTSON 1968 Down). The age of the G6PD A- allele was estimated from the decay of linkage disequilibrium and from coalescent simulations using the computer program GENETREE (HARDING et al. 1997 Down; BAHLO and GRIFFITHS 2000 Down). The SWST haplotype test of ANDOLFATTO et al. 1999 Down was implemented using the data from G6pd and L1cam separately. This test compares the observed number of haplotypes with those expected under a neutral model with a specified rate of recombination.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Nucleotide diversity:
Patterns of nucleotide variability at G6pd and L1cam are presented in Table 1 and Table 2. In the worldwide sample of 41 chromosomes (nonaugmented sample) we observed 18 single-nucleotide polymorphisms and three insertion/deletion (indel) polymorphisms at G6pd. Fifteen of these polymorphisms were in introns; of the remaining 6 polymorphisms, 2 were nonsynonymous changes (coding sites 202 and 376) and 4 were synonymous changes. Levels of nucleotide variability were roughly four times higher in Africa than in non-African populations (Table 2), consistent with other studies that demonstrate higher diversity in Africa (e.g., HARRIS and HEY 1999 Down, HARRIS and HEY 2001 Down; NACHMAN and CROWELL 2000 Down). Many of the polymorphisms found in Africa distinguish G6PD A- alleles from all other alleles in the sample. At L1cam we observed 7 polymorphisms in the nonaugmented sample. Levels of nucleotide variability were relatively low for L1cam overall; however, nucleotide variability was higher in Africa than in non-African populations.


 
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  		Table 2. Summary statistics of nucleotide variability for G6pd and L1cam

In the worldwide sample of 41 chromosomes, two A- alleles were in the African subset (n = 10), consistent with previously documented frequencies of ~20% for G6PD A- in sub-Saharan Africa. Overall, worldwide levels of nucleotide variability at G6pd and L1cam were close to or slightly below average values for other regions of the genome. For example, among primarily noncoding sites at 12 X-linked genes in humans, the average level of nucleotide diversity ({pi}) is 0.06% and the average proportion of segregating sites (Watterson's {Theta}) is 0.07% (NACHMAN 2001 Down). For both G6pd and L1cam, nucleotide diversity at intron sites is slightly below average (G6pd {pi} = 0.04%, L1cam {pi} = 0.02%), while Watterson's {Theta} is close to average (G6pd {Theta} = 0.08%, L1cam {Theta} = 0.07%). Since the A- allele represents only 5% of the worldwide sample, it is not expected to contribute substantially to levels of nucleotide variability. Within Africa, however, G6PD A- is present at high frequency (20%), yet overall levels of nucleotide variability ({pi} = 0.08%, Table 2) are still average. For example, the average level of nucleotide variability for 8 X-linked genes in Africa is 0.084% (PAYSEUR and NACHMAN 2002 Down).

Tests of neutrality:
Tajima's D is the normalized difference between {pi} and {Theta} and takes on positive values when there is an excess of intermediate-frequency polymorphisms and takes on negative values when there is an excess of low-frequency polymorphisms (TAJIMA 1989 Down). Positive Tajima's D values are generally consistent with long-term balancing selection or a population contraction, while negative values are expected following a selective sweep or a population expansion. For G6pd, Tajima's D is negative (but not significant) in the worldwide sample and for all subsets of the data (Table 2). Similar results are obtained with Fu and Li's D (FU and LI 1993 Down), which also measures the frequency distribution of polymorphisms and is sensitive to the number of singletons in the sample (Table 2). For L1cam both statistics are also negative and are significantly negative in the worldwide sample (Table 2). Fay and Wu's H statistic (FAY and WU 2000 Down) utilizes the frequency distribution of polymorphisms to test for an excess of high-frequency-derived variants compared to equilibrium neutral expectations. For both G6pd and L1cam, Fay and Wu's H test shows no significant deviation from the neutral expectation in the worldwide sample, the African sample, or the non-African sample.

We performed an HKA test (HUDSON et al. 1987 Down) using pairwise comparisons of polymorphism and divergence for G6pd and L1cam and two other X-linked genes, Pdha1 and Dmd. In comparisons using worldwide samples or African samples alone, we failed to reject the null model (HKA {chi}2 < 3.0, P > 0.1 for all tests). Thus, neither the frequency spectrum nor the level of heterozygosity at G6pd fits the expected pattern of nucleotide variability under a simple model of long-standing diversifying or balancing selection.

To test whether the haplotype structure of the data deviates from neutral expectations we implemented the SWST program as described in ANDOLFATTO et al. 1999 Down, assuming recombination rates of 0, 1, and 2 cM/Mb. Tests were performed separately for G6pd and for L1cam. None of these tests showed significant deviations from neutral expectations using the worldwide sample or the African sample alone.

Linkage disequilibrium:
To better examine patterns of linkage disequilibrium we augmented our random sample of 10 African X chromosomes with 4 chromosomes carrying A- alleles and 2 chromosomes carrying A+ alleles. Thus the augmented African sample in the study includes 6 chromosomes carrying G6pd A- alleles from South Africa, Central Africa, and West Africa (samples YCC 9, YCC 32, G11, M115, M241, and S823 in Table 1). Unusually high levels of linkage disequilibrium were observed within G6pd, within L1cam, and between G6pd and L1cam. D' is a measure of linkage disequilibrium that is standardized to equal 0 when there is random association among polymorphisms (i.e., no disequilibrium) and to equal 1 when there is complete association among polymorphisms (i.e., complete disequilibrium). In all comparisons between A- alleles and other alleles, D' = 1 for all sites in Table 1. A single most parsimonious haplotype network was inferred for all sites at G6pd (Fig 2), indicating a lack of evidence for recombination in this sample despite the fact that Xq28 is a genomic region with moderate to high rates of recombination (PAYSEUR and NACHMAN 2000 Down). Surprisingly, the three polymorphic sites at L1cam at intermediate frequency (positions 776, 885, and 2115) can also be mapped on this same network with no homoplasy. The observed high level of linkage disequilibrium is primarily a consequence of mutations falling on the branch separating the A- deficiency allele from the normal B alleles (Fig 2). A Fisher's exact test revealed significant LD (P = 0.0082) between site 202 of G6pd and three out of four informative polymorphisms at L1cam (alignment positions 776, 885, and 2115 at L1cam; Table 3).



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  		Figure 2. Haplotype network for polymorphisms of the worldwide sample at G6pd. Text inside circles represents sample identities (Table 1). Marks indicate polymorphisms and are labeled with the respective alignment position from Table 1. * indicates known haplotypes for G6PD A+ alleles that were not captured in this sample (VULLIAMY et al. 1991 Down). The solid circle represents G6PD A-, shaded circles represent G6PD A+, and open circles represent G6PD B alleles.


 
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  		Table 3. Nonrandom associations between G6pd site 202 and polymorphisms at L1cam

Age of the G6PD A- allele:
We estimated the age of the A- allele in two ways. First, we used a standard model for the decay of linkage disequilibrium as a function of time (t) and recombination (c), where linkage disequilibrium at time t (r2t) compared with time 0 (r20) is given by r2t/r20 = (1 - c)t (HEDRICK 1998 Down). For this calculation, we use r2 as a measure of linkage disequilibrium between L1cam and G6pd because, unlike D', r2 is sensitive to allele frequencies when only three out of four gametic types are present in a sample (HEDRICK 1998 Down). Assuming that linkage disequilibrium between site 202 of G6pd A- and positions 776, 885, and 2115 of L1cam alleles was complete at the time of origin of the A- allele (i.e., r20 = 1), we can estimate the time in generations, t = ln(r2t)/ln(1 - c), for the age of the allele, given the observed recombinational distance between L1cam and G6pd and the observed linkage disequilibrium in the data (r2 = 0.52). Since it is possible that r20 was <1.0 between these sites when the G6PD A- allele arose, our estimates provide an upper bound for the age of the G6PD A- allele. This region of Xq28 is subject to moderate levels of recombination in general (1–3 cM/Mb; PAYSEUR and NACHMAN 2000 Down; KONG et al. 2002 Down), and recombination rates near G6pd and L1cam specifically have been estimated as low as 0.14 cM/Mb and as high as 2 cM/Mb (SMALL et al. 1997 Down). Using these recombination rates we estimated the maximum age of the A- allele to be between 58 and 840 generations (Fig 3). With a generation time of 25 years, this implies that the G6pd A- allele arose 1461–20,994 years ago.



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  		Figure 3. Plot of linkage decay between G6pd (site 202) and L1cam (positions 776, 885, and 2115). The expected plot of linkage decay as measured by r2 is shown over time (t) for a range of two different recombination rates that have been suggested for the chromosomal region near G6pd and L1cam (SMALL et al. 1997 Down), 0.14 cM/Mb (long-dashed line) and 2 cM/Mb (solid line). The observed r2 = 0.52 provides minimum (tmin = 58 generations) and maximum (tmax = 840 generations) ages for the G6pd A- allele.

A second estimate for the age of the A- allele was obtained from simulations using a coalescent model conditioned on the sample size and observed levels of nucleotide variability (GENETREE: HARDING et al. 1997 Down; BAHLO and GRIFFITHS 2000 Down). This model assumes neutral equilibrium conditions and thus may provide an overestimate of the true age of the A- allele (since the present frequency of A- has probably been determined in large measure by selection). These simulations suggest that the A- allele arose 10,575 years ago (SD ± 8887 years).

Both of these estimates are in good agreement with an independent estimate for the age of the G6PD A- allele (3840–11,760 years) that was reported by TISHKOFF et al. 2001 Down on the basis of intra-allelic levels of linked microsatellite variability.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

Models of selection and nucleotide variability at G6pd:
We investigated levels and patterns of nucleotide variability at G6pd, a locus known to be under malarial selection in some human populations, and found that nucleotide diversity was similar to average values for other X-linked genes. Moreover, several commonly employed statistical tests based on DNA sequence variation failed to reject a simple neutral model of molecular evolution. In several respects, however, the data from G6pd are quite striking: levels of linkage disequilibrium are high and extend over a long genomic distance, much of the nucleotide variation is partitioned between functionally distinct alleles, and no nucleotide variation is observed within deficiency alleles. Below we discuss general models of selection for G6pd and how our observations might fit these models.

Although four different species of Plasmodium typically infect humans, P. falciparum is the most virulent species and is responsible for most malaria-related deaths, especially in Africa (SCHMIDT and ROBERTS 1996 Down). Malaria is endemic throughout most of sub-Saharan Africa where >1 million people die each year due to complications from infection (TRIGG and KONRACHINE 1998 Down). From a population genetics perspective, such a virulent parasite serves as a strong selective agent for genetic resistance. In fact, it has long been known that African populations exhibit genetic resistance factors to malaria at relatively high frequencies compared with non-African populations (e.g., MILLER 1994 Down). Moreover, many of the mutations that confer resistance are deleterious outside of the malaria environment. G6PD A-, for example, has an enzymatic activity that is only about one-tenth of normal and results in significant clinical manifestations such as hemolytic anemia and neonatal jaundice (HIRONO and BEUTLER 1988 Down; BEUTLER et al. 1989 Down; VULLIAMY et al. 1991 Down). However, this allele also confers an ~50% reduction in risk of severe malaria in both heterozygote females and hemizygote males (RUWENDE et al. 1995 Down).

Although G6PD is often assumed to be subject to balancing selection (sensu heterosis; e.g., TISHKOFF et al. 2001 Down), the precise nature of selection on G6PD deficiency alleles is not fully understood. In the absence of malaria, deficiency alleles are at a selective disadvantage and are expected to be eliminated (Table 4, fitness array 1). In the presence of malaria, female heterozygotes and male deficiency hemizygotes appear to have a selective advantage over wild-type individuals, but the fitness of female deficiency homozygotes relative to other genotypes is not clear (RUWENDE et al. 1995 Down). If female heterozygotes have a higher fitness than either homozygote, then selection may maintain both A- and wild-type alleles in populations under malarial selection (i.e., heterosis; Table 4, fitness array 2). However, the conditions for maintenance of a stable X-linked polymorphism are rather restrictive; either selection must be of similar magnitude but opposite in direction for the two sexes (which seems unlikely for G6PD deficiency) or there must be heterosis in females without a large fitness difference between the two male genotypes (HEDRICK 1998 Down). Alternatively, if female deficiency homozygotes have the same fitness as male hemizygotes and female heterozygotes, then selection should drive the eventual fixation of the G6PD A- allele in populations subject to continuous malarial selection (Table 4, fitness array 3). In such a situation, the A- allele is expected to rise to high frequencies and to reach fixation in a very short period of time (e.g., <10,000 years; RUWENDE et al. 1995 Down). The exact time required for fixation depends on assumptions about population size, initial frequency of the A- allele, relative fitness of the different genotypes, and the average generation time. However, for a wide range of parameter values, allele frequencies are expected to quickly rise to very high levels. The observation that most African populations have A- allele frequencies <20% (LIVINGSTONE 1985 Down) is inconsistent with a simple model of directional selection in which selection has been strong and long acting.


 
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  		Table 4. Fitness arrays under different malarial-selection regimes

Thus the best explanation for current G6PD A- allele frequencies seems to be either heterosis (fitness array 2) or some form of spatially and/or temporally varying selection due to malaria, in which case allele frequencies may be determined primarily by changing selection pressures (i.e., a combination over time or space of fitness array 1 and fitness array 2 and/or 3 in Table 4). On a large geographic scale (e.g., among continents), spatially varying selection is clearly important in determining allele frequencies; the extent to which this applies to small geographic scales is less clear, although the frequency of the A- allele differs significantly among different populations in sub-Saharan Africa (CAVALLI-SFORZA et al. 1996 Down; TISHKOFF et al. 2001 Down). While we cannot distinguish between heterosis and spatially/temporally varying selection, our data do allow us to address the timescale over which selection has acted.

A simple model of long-term balancing selection or long-term spatially or temporally varying selection is expected to leave a distinct signature in patterns of DNA sequence variation (Fig 4). When a new advantageous mutation first appears (Fig 4A), it will rise in frequency, creating LD with other mutations on the haplotype on which it arose (Fig 4B). This transient phase will result in lowered levels of heterozygosity. Over time, linkage disequilibrium will decay through recombination around the target of selection, and heterozygosity will increase near the target of selection (Fig 4C). This simple model of long-term selection predicts elevated levels of nucleotide variability in a restricted window around the target of selection (HUDSON et al. 1987 Down) and a skew in the frequency distribution of polymorphisms with an excess of intermediate-frequency variants within this restricted window (TAJIMA 1989 Down). Both of these patterns are seen in several other well-studied systems. For example, at Mhc loci in a variety of organisms [the human leukocyte antigen (HLA) loci in humans], levels of heterozygosity are significantly higher than those in neighboring regions (TAKAHATA et al. 1992 Down). At Adh in Drosophila melanogaster, heterozygosity is elevated around the fast/slow allozyme polymorphism, resulting in a significant HKA test (HUDSON et al. 1987 Down).



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  		Figure 4. Temporal schematic model for patterns of nucleotide variability at a locus under diversifying selection. Each group of 10 horizontal lines represents alleles sampled from a population at a given point in time. Vertical marks represent neutral polymorphisms. A red star represents the advantageous mutation under selection, and the ancestral allele bearing this mutation is marked by a red line. (a) A new advantageous mutation arises. (b) The new mutation quickly rises in frequency due to selection and is in linkage disequilibrium with neutral mutations over long distances. Heterozygosity is reduced relative to the population at the time (a). (c) Over time, for a locus under diversifying selection heterozygosity is elevated near the selected site, and linkage disequilibrium decays as a function of genetic distance.

In contrast, patterns of nucleotide variability at G6pd do not support either of these predictions with respect to G6pd A-, and several observations suggest that patterns at G6pd fit the model expected in an early stage of selection (Fig 4B). First, overall levels of nucleotide diversity are close to average values for other X-linked loci. This is true for the worldwide sample and, more importantly for evaluating models of selection, it is also true for the African sample alone. An HKA test applied to our data fails to reject a neutral model. Second, there is no evidence for an excess of intermediate-frequency polymorphisms. In fact, both Tajima's D and Fu and Li's D are slightly (but not significantly) negative for the African sample (Table 2). Third, we find extensive linkage disequilibrium within and around G6pd, and this disequilibrium is due almost exclusively to nucleotide differences that distinguish the A- allele from other alleles. We observed no recombination events within G6pd. This stands in contrast to many other human nucleotide polymorphism data sets, including intron 44 of Dmd, surveyed in this same set of individuals (NACHMAN and CROWELL 2000 Down), in which numerous recombination events were observed over distances of several hundred bases. In addition to significant LD within G6pd, we found significant LD between G6pd and L1cam (Table 3; D' = 1 in all comparisons), loci that are separated by ~550 kb. This amount of LD is much higher than typical values for the human genome. For example, REICH et al. 2001 Down recently studied the decay of D' for 19 different genomic regions and found that in a European population the half-length of D' (the distance at which the average D' drops below 0.5) is typically 60 kb, while in an African population the half-length of D' is <5 kb (REICH et al. 2001 Down). Other studies have also revealed lower levels of linkage disequilibrium in African populations compared with non-African populations (TISHKOFF et al. 1996 Down, TISHKOFF et al. 2001 Down). Interestingly, we observe much higher levels of LD than those previously reported for this region of Xq28 by TAILLON-MILLER et al. 2000 Down in populations of European descent. Finally, there is no intra-allelic variation within G6pd A-, consistent with the notion that G6PD A- is relatively young.

Taken together, these observations argue against a model of long-term selection on the G6pd A- allele, but do not allow us to distinguish between recent balancing selection (sensu heterosis), on the one hand, and recent diversity-enhancing (i.e., spatially and/or temporally varying) selection, on the other hand. Better fitness estimates of all genotypes (in particular, female deficiency homozygotes), as well as detailed sampling of G6PD A- frequencies across Africa, might help us to distinguish between these hypotheses.

Contrary to the intra-allelic patterns of nucleotide variability for G6pd A-, the minor deficiency allele G6pd A+ shows a high level of intra-allelic diversity and greater linkage equilibrium. Although our study includes only two A+ chromosomes that represent a single haplotype, at least two additional haplotypes have been identified on the basis of RFLP analyses (Fig 2; VULLIAMMY et al. 1991). Moreover, microsatellites located up to 19 kb away from G6pd exhibit greater linkage equilibrium and higher diversity on A+ alleles than on A- alleles (TISHKOFF et al. 2001 Down). These observations, taken together with a coalescent-based estimate for the age of the mutation at coding position 376 from our study (131,250–174,375 years on the basis of GENETREE analysis), suggest that G6PD A+ may be relatively old. G6PD A+ has an enzymatic activity that is 80% of normal and does not appear to cause a significant clinical condition (TAKIZAWA et al. 1987 Down). Furthermore, G6PD A+ does not seem to currently confer resistance to severe falciparum malaria, as does G6PD A- (RUWENDE et al. 1995 Down). However, the age of G6PD A+ coupled with the reduced level of enzymatic activity raises the possibility that this allele has been under selection at some time in the past.

Is it possible that demographic processes are primarily responsible for the high levels of LD seen in Fig 2? Linguistic and archaeological evidence suggests that a Bantu expansion took place in Africa ~4000 years ago (EXCOFFIER et al. 1987 Down). This range expansion occurred in sub-Saharan Africa primarily from west to east and southward, a distribution that is similar to the current distribution of African populations with elevated G6PD- allele frequencies. If admixture from this range expansion were responsible for generating the observed LD in our data, we would also expect to see G6PD B alleles with significant LD. This is not observed. Instead, most of the LD in our data is found between sites on functionally different alleles, arguing against any simple demographic explanation. Likewise, no LD is observed between Bantu and non-Bantu individuals from this set of 41 individuals sampled for other loci (e.g., NACHMAN and CROWELL 2000 Down).

One intriguing observation in our data set is the relatively high level of divergence found at L1cam between individuals bearing the G6PD A- allele and all other individuals. Four of the six (66.7%) G6PD A- alleles share a common motif of three polymorphisms in complete linkage disequilibrium (C, T, and T at positions 776, 885, and 2115, respectively; Table 1) while the rest of the segregating sites at L1cam include only four singletons and one doubleton. This pattern along with the significant LD between G6pd and L1cam (Table 3) suggests that the A- mutation arose on a relatively diverged haplotype, possibly as a consequence of population subdivision. Analysis of G6pd and L1cam as well as additional neighboring loci in a larger geographic sample from Africa may shed light on this unusual pattern.

In general, the observations reported here demonstrate that even when selection is relatively strong, its signature on patterns of DNA sequence variation may be subtle, especially if selection is recent. While several of the conventional statistical tests for selection fail to reject the null hypothesis, the footprint of selection is seen in the long-range patterns of LD and in the absence of variation among A- alleles from different parts of Africa. Similar patterns of nucleotide variability at G6pd have also recently been reported by VERRELLI et al. 2002 Down. The patterns of DNA sequence variation observed at G6pd are markedly different from those seen at another well-studied target of balancing selection, HLA, where ancient alleles result in substantially elevated levels of polymorphism (GRIMSLEY et al. 1998 Down; HORTON et al. 1998 Down; GAUDIERI et al. 1999 Down). The spatial and temporal scales over which selection pressures have shaped human genomic diversity are still largely unknown, but environmental changes associated with the transition from the Paleolithic to the Neolithic may have imposed substantial new selection pressures on humans, suggesting that patterns of nucleotide variability similar to those documented here for G6pd may be found at other loci.

Age of G6PD A- and the evolution of malarial resistance:
These results have important implications for the evolution of resistance to malaria in humans. Several observations reported here, including average levels of nucleotide variability at G6pd, negative values of Tajima's D, high levels of linkage disequilibrium between G6pd and L1cam, and complete absence of variation among G6pd A- alleles from different parts of Africa, suggest that the A- allele is young (Table 1 and Table 2). A recent study based on microsatellite haplotype diversity (TISHKOFF et al. 2001 Down) also suggests that the G6pd A- allele arose recently, within the past 4000–12,000 years. A recent phylogeny of primate malaria parasites indicates that P. falciparum is closely related to P. reichenowi, a chimpanzee parasite. Moreover, cytochrome b sequence divergence between P. falciparum and P. reichenowi suggests a divergence time of 4–5 million years ago (ESCALANTE et al. 1998 Down), in good agreement with the estimated time of the human-chimpanzee divergence. The discrepancy between this date and the recent origin of the G6pd A- allele raises the possibility that P. falciparum has been a human parasite for most of the evolutionary history of H. sapiens, but that the parasite's current level of virulence has evolved only recently (RICH et al. 1998 Down). The estimated age of the A- allele agrees well with the spread of agriculture throughout sub-Saharan Africa (WATERS et al. 1991 Down; CAVALLI-SFORZA et al. 1996 Down) and suggests that changes in human lifeway may have contributed to increased transmission and/or increased virulence of P. falciparum, perhaps through an increase in the density and mobility of Anopheles mosquitoes that serve as vectors in transmission of malaria.


*  ACKNOWLEDGMENTS

We thank J. D. Jensen and S. Peterson for technical assistance. Human DNA samples M115 and M241 were kindly donated by L. Luzzatto and K. Nafa. R. O. Ryder provided chimpanzee and orangutan samples. R. M. Harding, L. Luzzatto, E. Beutler, B. A. Payseur, E. T. Wood, C. C. Campbell, and A. J. Redd provided helpful discussion. We also thank R. G. Harrison and two anonymous reviewers who provided helpful comments about the manuscript. This work was supported by a National Science Foundation (NSF) grant to M.W.N. and M.F.H. and an NSF predoctoral fellowship to M.A.S.

Manuscript received January 14, 2002; Accepted for publication September 18, 2002.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
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