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Kouprina et al, 2004 wrote:Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.

Kouprina et al, 2004 wrote:Introduction

The human brain, particularly the cerebral cortex, has undergone a dramatic increase in its volume during the course of primate evolution, but the underlying molecular mechanisms that caused this expansion are not known. One approach shedding light on the molecular mechanisms of brain evolution is the analysis of the gene mutations that lead to defects in brain development. Among the best examples of such defects is the human primary microcephaly syndrome. Primary microcephaly (MCPH) is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture (McCreary et al. 1996; Mochida and Walsh 2001). Moreover, there are no recognizable abnormalities in the organs other than the central nervous system. The most common cause of MCPH appears to be mutations in the ASPM gene (Roberts et al. 2002).

The ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats (calmodulin-binding motifs), and a C-terminal region (Bond et al. 2002). Though the exact function of the human ASPM in the brain needs to be clarified, the homologue in the fruit fly, Drosophila melanogaster, abnormal spindle (asp), is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development (Ripoll et al. 1985; do Carmo Avides et al. 2001; Riparbelli et al. 2001). In the mouse (Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis (Bond et al. 2002). This expression profile suggests a potential role for Aspm in regulating neurogenesis.

Interspecies comparisons of ASPM orthologs have shown their overall conservation, but also a consistent correlation of greater protein size with larger brain size (Bond et al. 2002). The increase in protein size across species is due mainly to the increased number of IQ repeats, suggesting that specific changes in ASPM may be critical for evolution of the central nervous system.

In an attempt to reconstruct the evolutionary history of the ASPM gene, we isolated large genomic clones containing the entire ASPM gene in several nonhuman primate species. Sequence analysis of these clones revealed a high conservation in both coding and noncoding regions, and showed that evolution of the ASPM gene might have been under positive selection in hominoids. These clones could also provide important reagents for the future study of ASPM gene regulation in its native sequence context.

Kouprina et al, 2004 wrote:Results

Comparison of Genomic Organization of the ASPM Genes in Primates

Homologues from chimpanzee (Pan troglodytes), gorilla ([/i]Gorilla gorilla[/i]), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library (Kouprina and Larionov 2003). The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast. Since up to 15% divergence in DNA sequences does not prevent selective gene isolation by in vivo recombination in yeast (Noskov et al. 2003), for cloning purposes, a TAR vector was designed containing short human ASPM-gene-specific targeting hooks specific to the exon 1 and 39 noncoding regions (see ‘‘Materials and Methods’’). The TAR cloning scheme for isolating the ASPM gene homologues from nonhuman primates is shown in Figure 1. The yield of ASPM-positive clones from chimpanzee, gorilla, orangutan, and rhesus macaque was the same as that from the human DNA, suggesting that most homologous regions from nonhuman primates can be efficiently cloned by in vivo recombination in yeast using targeting hooks developed from human sequences.

We have compared complete gene sequences from primate species with a 65-kb, full-size human ASPM gene. All the analyzed genes are organized into 28 exons encoding a 3,470–3,479-amino-acid-long protein. ASPM genes start with an approximately 800-bp-long CpG island, that harbors promoter sequences, 59 untranslated regions, and the first exon (Figure 2). ASPM sequences share a high degree of conservation (Figure 2H), and pairwise DNA identity ranges from 94.5% for macaque and gorilla to 99.3% for the human–chimpanzee comparison (Table 1). Multiple alignment of the genes revealed a low proportion of indels. Only ten insertions/deletions equal to or longer than 50 bp have been found, all of them located within introns (Figure 2B). Seven detected insertions were mainly associated with repetitive DNA: two (AT)n microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2. Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown).

Of the two detected deletions, the macaque-specific 72-bp long deletion appears to be associated with nonrepetitve DNA. The second one, an 818-bp-long deletion in orangutan, was probably caused by homologous Alu–Alu recombination (see below and Figure S1). The remaining indels are related to expansion/contraction of a short minisatellite array. It was caused either by a 53-bp expansion in the gorilla–chimpanzee–human clade or by two independent deletions/contractions in the macaque and orangutan lineages.

An approximately 3-kb-long intronic segment between exons 4 and 5 is present in several copies in the genome (Figure 2E; Figure S2). Closer analysis of the human genome confirmed that copies of this region are homologous to 24 segmental duplications located mainly in telomeric regions of Chromosomes 1–8, 10, 11, 16, 19, 20, and Y. Based on the sequence similarity and the presence of an L1P4 LINE insertion at the 5' end, the most closely related are three duplications at 7q11–13. The most similar copy is located on Chromosome 7 and shares 93% identity with the ASPM intronic segment. Five duplications are located on Chromosome 1; the closest copy is found 27 Mb away from the ASPM gene.

We looked for several common motifs associated with genomic breakpoints in cancers (Abeysinghe et al. 2003). Figure 2F shows the positions of such potentially unstable oligonucleotides. Interestingly, the orangutan-specific deletion (Figure 2B) has its 5' breakpoint located just 1 bp upstream of a sequence 100% identical to the chi-like consensus motif GCWGGWGG (see Figure S1). The chi motif is recognized by the RecBCD-mediated recombination pathway in prokaryotes and seems to be associated with rearrangements in the human genome (Dewyse and Bradley 1991; Chuzhanova et al. 2003). Both deletion breakpoints in the orangutan deletion are located within 5' parts of two Alu sequences, suggesting that the deletion was created by homologous Alu–Alu recombination. Similar homologous recombinations with breakpoints located near chi-like motifs in 5' regions of Alu sequences were described previously (Chen et al. 1989; Rudiger et al. 1995).

In summary, despite the presence of a few indels, coding and noncoding regions of ASPM homologues show a marked degree of conservation.

Kouprina et al, 2004 wrote:Evolution of the ASPM Protein

We have analyzed ASPM CDSs from six primate species: human, chimpanzee, gorilla, orangutan, rhesus macaque, and African green monkey (Cercopithecus aethiops). Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure (Figure 3A). The IQ repeat domain contains the same number of repeats, suggesting that their expansion occurred in early primate evolution. The CDSs are, as expected, more conserved than the complete gene sequences with promoter and intronic regions (Table 2; Table 3). Only six short indels were identified (Figure 3B).

From the DNA and protein conservation profiles (Figure 3I), ASPM segments evolve differently along the length of the CDS. N- and C-terminal regions and the region corresponding to exons 5–15 are conserved. In contrast, exons 3 and 4 and the complete IQ repeat domain (positions 1,267–3,225) are more variable. Indeed, nonsynonymous substitutions in hominoid primates (Figure 3C) and in ancestral lineages (Figure 3D) and nonsynonymous polymorphism (Figure 3E) are nearly absent in the conserved central (exons 5–15) and C-terminal regions. This pattern indicates different rates of evolution along the ASPM protein, visualized by plots of synonymous Ks and nonsynonymous Ka rates (Figure 3H) and supported by phylogenetic analysis (see below and Figure 4). It is notable that the comparison of the primate and mouse proteins also revealed the same pattern of conservative and nonconservative regions along ASPM protein (Figure S3).

Analysis of the nonsynonymous/synonymous substitution ratio (x = Ka/Ks) revealed an elevated value in the human branch (Figure 4A). According to the likelihood ratio test, the human x rate is significantly different from the rate in the rest of the tree (p < 0.05). Also the model that the complete gorilla–chimpanzee–human clade is evolving at one x rate different from that in the rest of the tree is well supported (p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation (see Figure 3), we separately analyzed a conserved region (exons 5–15 plus a small part of exon 16) and a variable IQ repeat domain. As can be seen (Figure 4B) the conserved region has all branches shorter, indicating overall a slower rate of evolution. In the human lineage, the x ratio equals zero; however, the test for whether the human branch has a different (lower) x rate than the rest did not yield significant values. In contrast, the tree based on the variable IQ repeat domain exhibits x values greater than one for the human and gorilla branches (Figure 4C). The likelihood ratio test supports the model in which human and gorilla lineages evolved under a significantly higher x ratio than the rest of the tree. Similar results were obtained for exon 18 with additional sequences from two New World monkeys (Figure 4D). As seen from Figure 4A–4D, different sequences from African green monkey, gorilla, and chimpanzee individuals result in different x values for their corresponding terminal branches. One chimpanzee sequence also produced an x ratio greater than one for exon 18 (Figure 4D). It is worth noting that neither codon bias nor selection on third codon positions seemed to influence the synonymous rate Ks strongly (Table S1). Therefore, the high Ka/Ks ratios in human and gorilla are likely to be products of adaptive evolution.

Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for ASPM polymorphism in those species (see Figure 3E). Human polymorphism data from ASPM mutant haplotypes are not representative of wild-type variation so were not used in these comparisons. For African green monkey, five synonymous and five nonsynonymous changes were found between two sequences. The gorilla and chimpanzee CDSs in particular showed an apparently high degree of replacement polymorphism. Gorilla polymorphism included 35 point mutations (15 silent mutations and 21 replacements). Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test (McDonald and Kreitman 1991). In the case of chimpanzee polymorphism compared to divergence with human, we could not reject the null hypothesis that polymorphism and divergence between species were significantly different (William’s adjusted G statistic = 0.083, chi-square with 1 d.f., not significant; values based on PAML-generated Ka and Ks values using the free ratio model). Gorilla polymorphism was compared to divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. In this case we can reject the null hypothesis (William’s adjusted G statistic = 122.45, chi-square with 1 d.f., p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the x ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM.

Kouprina et al, 2004 wrote:Discussion

In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns.

Analysis of nonsynonymous/synonymous substitution ratios indicates different rates of evolution along the ASPM protein: part of ASPM evolved under positive selection while other parts were under negative (purifying) selection in human and African ape lineages. Such ‘‘mosaic’’ selection has been previously described for other proteins (Endo et al. 1996; Crandall et al. 1999; Hughes 1999; Kreitman and Comeron 1999). When our work was completed, the paper by Zhang supporting accelerated evolution of the human ASPM was issued (Zhang 2003). However, because the author did not analyze the gorilla gene homologue, he concluded that accelerated sequence evolution is specific to the hominid lineage. Our finding that selection on ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular ‘‘exaptation’’ (Gould and Vrba 1982), in that the originally selected function of ASPM was for something other than large brain size.

In the case of ASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats (Bond et al. 2002). For example, the asp homologue of the nematode Caenorhabditis elegans contains two IQ repeats, the fruit fly—24 IQ repeats, and the mouse—61 IQ repeats, and there are 74 IQ repeats in humans (Bond et al. 2002). ASPM homologues in the nonhuman primates examined here contain the same number of IQ repeats as human, supporting the idea that repeat expansion occurred prior to the anthropoid divergence (which gave rise to New World monkeys, Old World monkeys, and hominoids) and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution.

Human ASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders (Clark et al 2003). However, ASPM is unique because its distinctive pattern of accelerated protein evolution begins several million years prior to brain expansion in the hominid lineage. Absolute brain size in orangutans (430 g in males; 370 g in females) is barely different from that in gorillas (530 g in males; 460 g in females) and common chimpanzees (400 g in males; 370 g in females) (Tobias 1971), yet accelerated ASPM evolution began in the common ancestor of gorillas, chimpanzees, and humans, approximately 7–8 million years ago. Only much later did brain expansion begin in hominids, starting at 400–450 g roughly 2–2.5 million years ago and growing to its final current size of 1350–1450 g approximately 200,000–400,000 years ago (Wood and Collard 1999). Therefore genotypic changes in ASPM preceded marked phenotypic changes in hominoid brain evolution, at least at the level at which they have currently been studied. The molecular changes in ASPM may predict the existence of differences in early neurogenesis between orangutans, on the one hand, and gorillas, chimpanzees, and humans, on the other, which may manifest as more subtle differences in brain anatomy than gross changes in brain volume.

How might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically ‘‘symmetric’’ in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically ‘‘asymmetric’’ so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size (Chenn and Walsh 2002), and so changes in ASPM could regulate cortical size by making subtle changes in spindle orientation. Alternatively, evolutionary changes in ASPM may not themselves have led to increase in the size of the brain, but instead perhaps ASPM might be essential to insure faithful DNA replication and proper chromosome segregation. In rodents, a surprising number of cerebral cortical neurons are aneuploid (Rehen et al. 2001). Perhaps directed selection of specific domains of ASPM helps insure faithful chromosome segregation to allow a larger number of cerebral cortical neurons to be formed without an unduly high incidence of chromosome aneuploidy.

Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits.

Note

The related paper by Evans et al. (2004) was published in Human Molecular Genetics shortly after this paper was submitted.

Ziang, 2003 wrote:ABSTRACT

The size of human brain tripled over a period of
2 million years (MY) that ended 0.2–0.4 MY ago. This evolutionary expansion is believed to be important to the emergence of human language and other high-order cognitive functions, yet its genetic basis remains unknown. An evolutionary analysis of genes controlling brain development may shed light on it. ASPM (abnormal spindle-like microcephaly associated) is one of such genes, as nonsense mutations lead to primary microcephaly, a human disease characterized by a 70% reduction in brain size. Here I provide evidence suggesting that human ASPM went through an episode of accelerated sequence evolution by positive Darwinian selection after the split of humans and chimpanzees but before the separation of modern non-Africans from Africans. Because positive selection acts on a gene only when the gene function is altered and the organismal fitness is increased, my results suggest that adaptive functional modifications occurred in human ASPM and that it may be a major genetic component underlying the evolution of the human brain.

Ziang, 2003 wrote:AMONG mammals, humans have an exceptionally big brain relative to their body size. For example, in comparison with chimpanzees, the brain weight of humans is 250% greater while the body is only 20% heavier (McHenry 1994). The dramatic evolutionary expansion of the human brain started from an average brain weight of 400–450 g ~2–2.5 million years (MY) ago and ended with a weight of ~1350–1450 g ~0.2–0.4 MY ago (McHenry 1994; Wood and Collard 1999). This process represents one of the most rapid morphological changes in evolution. It is generally believed that the brain expansion set the stage for the emergence of human language and other high-order cognitive functions and that it was caused by adaptive selection (Decan 1992), yet the genetic basis of the expansion remains elusive. A study of human mutations that result in unusally small brains may help identify the genetic modifications that contributed to the human brain expansion. In this regard, primary microcephaly (small head) is of particular interest (Mochida and Walsh 2001; Bond et al. 2002; Kumar et al. 2002). Microcephaly is an autosomal recessive genetic disease with an incidence of 4–40 per million live births in western countries (Mochida and Walsh 2001; Kumar et al. 2002). It is defined as a head circumference >3 standard deviations below the population age-related mean, but with no associated malfunctions other than mild-to-moderate mental retardation (Mochida and Walsh 2001; Kumar et al. 2002).

The reduction in head circumference correlates with a markedly reduced brain size. Microcephaly is genetically heterogeneous, associated with mutations in at least five loci (Mochida and Walsh 2001; Kumar et al. 2002), one of which was recently identified and named ASPM (abnormal spindle-like microcephaly associated; Bond et al. 2002). Four different homozygous mutations in ASPM introducing premature stop codons were found to cosegregate with the disease in four respective families, while none of these mutations were found in 200 normal human chromosomes (Bond et al. 2002). Because the brain size of a typical microcephaly patient (430 g; Mochida and Walsh 2001; Kumar et al. 2002) is comparable with those of early hominids such as the 2.3- to 3.0-MY-old Australopithecus africanus (420 g; McHenry 1994; Wood and Collard 1999), I hypothesize that ASPM may be one of the genetic components underlying the human brain expansion. Signatures of accelerated evolution of ASPM under positive selection during human origins would strongly support my hypothesis, because the action of positive selection indicates a modification in gene function resulting in elevated organismal fitness (Zhang et al. 2002). Below I provide population genetic and molecular evolutionary evidence for the operation of such adaptive selection on ASPM.

Ziang, 2003 wrote:RESULTS

Elevation of dN/dS in the human ASPM lineage:

Human ASPM has 28 coding exons, spanning 62 kb in chromosome 1p31 and encoding a huge protein of 3477 amino acids (Figure 1). I determined the entire coding sequences of ASPM from one human, one chimpanzee, and one orangutan, and compared them in the phylogenetic tree of the three species (Figure 2). The orangutan sequence is used as the outgroup for humans and chimpanzees so that nucleotide substitutions on the human and chimpanzee lineages can be separated. I did not sequence the gorilla because the gorilla sequence may not be appropriate as the outgroup due to incomplete lineage sorting (Satta et al. 2000). Use of orangutan, a slightly more distant outgroup, solves this problem. A commonly used indicator of natural selection at the DNA sequence level is the ratio of the rate of nonsynonymous nucleotide substitution (dN) to that of synonymous substitution (dS). Most functional genes show dN/dS < 1, because a substantial proportion of nonsynonymous mutations are deleterious and are removed by purifying selection, whereas synonymous mutations are more or less neutral and are generally uninfluenced by selection. A gene may occasionally exhibit dN/dS > 1 when a large fraction of nonsynonymous mutations are advantageous and are driven to fixation by positive selection (Li 1997; Nei and Kumar 2000). I estimated the dN/dS ratio for ASPM in each of the three tree branches (Figure 2), using a maximum-likelihood method, and found that dN/dS is lowest in the orangutan branch (0.43), higher in the chimpanzee branch (0.66), and highest in the human branch (1.03). The hypothesis of dN/dS = 1 is rejected for the orangutan branch (P < 0.001, likelihood-ratio test), but not for the other two branches, suggesting a difference in selection has occurred. Indeed, a test of the difference in dN/dS between the human and orangutan branches gives a marginally significant result (P = 0.064), but the difference between the chimpanzee and orangutan branches is not significant (P = 0.29), nor is the difference between human and chimpanzee branches (P = 0.45). Because the dN/dS ratio between the orangutan and mouse (Mus musculus) is also low (0.29), an increase of dN/dS in humans is more likely than a decrease in orangutans. The mouse sequence (GenBank accession no. AF533752) was not included in the phylogeny-based analysis as it is relatively distantly related to the ape sequences and contains multiple insertions and deletions, which would make the inference less reliable. Similar results are obtained when I first infer the ASPM sequence for the common ancestor of humans and chimpanzees and then estimate the dN/dS ratio by counting the numbers of synonymous and non- synonymous nucleotide substitutions on each branch. For instance, this approach gives dN/dS = 1.13, 0.84, and 0.52, respectively, for the human, chimpanzee, and orangutan branches.

Complete functional relaxation does not adequately explain the elevation of dN/dS:

Two hypotheses may explain the increase in dN/dS to 1.03 during the evolution of human ASPM. First, the functional constraints and purifying selection on ASPM may have been completely relaxed and many deleterious nonsynonymous mutations were fixed by random genetic drift. Alternatively, advantageous nonsynonymous substitutions under positive selection occurred at some sites, while purifying selection acted at some other sites, resulting in an average dN/dS of ~ 1. Under the first hypothesis, ASPM has been under pure neutral evolution since the human-chimpanzee separation ~6–7 MY ago (Brunet et al. 2002). Using rates of single-nucleotide mutations and insertion/deletion mutations estimated from human-chimpanzee genomic comparisons (Britten 2002; Yi et al. 2002), I conducted a computer simulation of neutral evolution of ASPM (see materials and methods). I found that the probability that ASPM retains its open reading frame after 6 MY of neutral evolution is extremely low (1.7 × 10-4). Even when the above two mutation rates are both halved, the probability is still very small (0.014), suggesting that ASPM must have been under purifying selection. The fact that nonsense mutations in ASPM lead to microcephaly also demonstrates the presence of functional constraints on the gene. Thus, the hypothesis of complete relaxation of functional constraints and lack of purifying selection for the past 6–7 MY of human evolution is inconsistent with the data, and some sites in ASPM must have been subject to purifying selection (dN/dS < 1). This result would imply, although not prove, that some other sites are under positive selection (dN/dS > 1), so that the average dN/dS across the entire protein is ~1. However, it is difficult to rule out the possibility of an incomplete functional relaxation in human ASPM, which can lead to a dN/dS ratio of ~1 when the number of substitutions is relatively small. A population genetic study may help resolve this question.

Signatures of purifying selection from population genetic data:

The entire coding sequence of ASPM is determined from 14 human individuals of different geographic origins. A total of 33 single-nucleotide polymorphisms are found (Tables 1 and 2). The derived and ancestral alleles are inferred using the chimpanzee and orangutan sequences as outgroups. Tajima’s (1989) and Fu and Li’s (1993) tests reveal slight departure of the data from the Wright-Fisher model of neutrality (D = -1.29, P =0.081; F = -1.76, P = 0.074; Table 2). But Fay and Wu’s (2000) test, which is designed to detect recent selective sweeps, does not show a significant result (H = -2.08, P = 0.21). Thus, the negative D and F likely reflect recent population expansions and/or purifying background selection. A recent study suggested that negative D values may also be found under certain sampling schemes if there is fine-scale population differentiation (Ptak and Przeworski 2002). When the synonymous and nonsynonymous sites were analyzed separately, I detected significant negative D and F values at nonsynonymous sites (P < 0.05; Table 2), but not at synonymous sites. H is not significant at either type of site. These results suggest that the nonsynonymous sites in human ASPM are subject to purifying selection. It should be mentioned that the recombination rate in the ASPM region is ~1.8 cM/106 nucleotides (Kong et al. 2002), which translates into 1.1 × 10-3 recombination/meiosis for the sequences analyzed here. This relatively high recombination rate localizes signatures of selection to a small region surrounding the selected sites. This might in part explain the above differences in the test results between synonymous and nonsynonymous sites.

Population genetic theory predicts that deleterious mutations do not reach high frequencies in populations, while neutral and advantageous mutations do. A comparison between rare and common polymorphisms may detect purifying selection of deleterious mutations (Fay et al. 2001). Fay et al. recommended a frequency of ~10% for the derived allele as a cutoff between rare and common polymorphisms (Fay et al. 2001, 2002). In the present sample of 28 chromosomes, derived alleles that appear one or two times are regarded as rare polymorphisms, and the rest are common. Because of the limited sample size, a truly rare allele may inadvertently appear more than twice in our sample and a truly common allele may inadvertently be regarded as rare. Using probability theory, I computed that the probability of the former error is <5% for an allele with frequency <3% and the probability of the latter error is <5% for an allele with frequency >20%. Thus, the present classification of rare and common alleles is expected to be relatively accurate. I observed that nR =15 nonsynonymous and sR = 5 synonymous rare polymorphisms and nC = 5 nonsynonymous and sC = 8 synonymous common polymorphisms from the present data (Table 2; Figure 1). The ratio of nC to nR (5/15 = 0.333) is significantly lower than that of sC to sR (8/5 = 1.6; [cvhr]967[/chr]2 = 4.41, P < 0.05; Table 2). Since synonymous mutations are more or less neutral, the observed deficit of common nonsynonymous polymorphisms suggests that purifying selection has prevented the spread of nonsynonymous deleterious mutations. It is estimated by the likelihood method that there areN=7459 and S=2972 potentially nonsynonymous and synonymous sites in ASPM, respectively. Thus, for rare polymorphisms, there are nR/N = 15/7459 = 2.01 × 10-3 polymorphisms/nonsynonymous site and sR/S = 5/2972 = 1.68 × 10-3/synonymous site. Their difference is statistically insignificant (χ2 = 0.09, P > 0.5). In contrast, for common polymorphisms, the number is significantly smaller per nonsynonymous site (nC/N = 5/7459 = 0.67 × 10-3) than per synonymous site (sC/S = 8/2972 = 2.69 × 10-3; χ2 = 6.98, P < 0.01), confirming that purifying selection has reduced the number of common nonsynonymous polymorphisms. This result also suggests the absence or rareness of advantageous nonsynonymous polymorphisms of ASPM that are currently segregating in humans, as such polymorphisms would predominantly show up as common polymorphisms and render nC/N higher. This is consistent with the above result from Fay and Wu’s test. The proportion of nonsynonymous polymorphisms not under purifying selection may be estimated by (nC/N)/(sC/S) = (0.67 × 10-3)/(2.69 × 10-3) = 0.25 or by (nC/sC)/(nR/sR) = (5/8)/(15/5) = 0.21. The two estimates are close to each other and to the dN/dS ratio between the mouse and orangutan (0.29). This indicates that human ASPM is currently under relatively strong purifying selection, and the strength of selection is comparable to or even greater than that in the long-term evolution of mammalian ASPM.

Comparison of polymorphism and divergence suggests past positive selection:

Because both the synonymous and nonsynonymous common polymorphisms are largely neutral, comparing them with the fixed substitutions between humans and chimpanzees can reveal the signature of selection that has influenced the substitution processes (McDonald and Kreitman 1991; Fay et al. 2001, 2002; Smith and Eyre-Walker 2002). This comparison shows a significant excess of fixed nonsynonymous substitutions (χ2 = 3.88, P < 0.05, Table 2), suggesting that some nonsynonymous substitutions were fixed by positive selection. Because the expansion of brain size occurred in the human lineage after the human-chimpanzee split, it is more relevant to examine whether the human branch exhibits an excess of nonsynonymous substitutions. For this, the ASPM sequence of the common ancestor of humans and chimpanzees was inferred by the Bayesian method. Because the sequences considered are closely related, this inference is reliable, with the average posterior probability >0.999. Comparing the ancestral sequence with the polymorphic human sequences, I identified 16 nonsynonymous and 6 synonymous mutations that have been fixed in the human lineage (Table 2; Figure 1). Their ratio (16/6 = 2.67) is significantly greater than that for common polymorphisms (nC/sC = 5/8 = 0.63; χ2 = 4.00, P < 0.05). The number of neutral nonsynonymous substitutions may be estimated from the number of synonymous substitutions multiplied by nC/sC, which yielded 6 × (5/8) = 3.75 (Fay et al. 2001, 2002; Smith and Eyre-Walker 2002). The number of nonsynonymous substitutions unexplainable by neutral evolution is 16 - 3.75 = 12, which may have been fixed by positive selection. It should be noted that a recent population expansion can cause an overestimate of the number of adaptive substitutions when slightly deleterious mutations are present. However, such overestimation is unlikely in the present case because the current effective population size of humans, even after the recent expansion, is still smaller than the long-term effective population size separating humans and chimpanzees and the effective population size of the common ancestor of humans and chimpanzees (Takahata et al. 1995; Chen and Li 2001; Kaessmann et al. 2001; Eyre-Walker 2002). It is interesting that there is no significant excess of nonsynonymous substitutions for either the chimpanzee or orangutan branches when the common polymorphisms and substitutions are compared (P > 0.05).

IQ repeats and brain size variation:

Human ASPM contains multiple calmodulin-binding IQ repeats (Bond et al. 2002). In a comparison of putative orthologous ASPM genes from the human, mouse, fruit fly (Drosophila melanogaster), and nematode (Caenorhabditis elegans), Bond et al. (2002) noticed that organisms with larger brains have more IQ repeats, implying a possible relation of IQ repeats and brain size. In particular, the predominant difference between the human and mouse ASPM genes is a large IQ-repeat-encoding insertion of 867 nucleotides at the end of exon 18. However, my data showed no difference in the number of IQ repeats between human and chimpanzee ASPM sequences. To trace the origin of the large insertion in human ASPM, I amplified and sequenced from several mammals two DNA segments that cover most of the insertion (Figure 1). Segment I is of 212 nucleotides and segment II is of 706 nucleotides. One or both segments were obtained from species belonging to primates, Cetartiodactyla, Carnivora, and Hyracoidea, but not from mouse or hamster (Figure 3). Phylogenetic analyses were conducted to confirm that the obtained sequences are orthologous to the human sequence (Figure 4). While nonamplification of a sequence does not prove its nonexistence, the amplification of the orthologous sequence indicates its presence. From the recently established mammalian phylogeny (Murphy et al. 2001), it can be inferred that the large human insertion was already present in the common ancestor of most placental mammals, but was deleted in mouse and possibly in other rodents (Figure 3). Thus, this IQ-repeat-containing sequence does not explain the brain size variation among many nonrodent mammals.

Ziang, 2003 wrote:DISCUSSION

In the above, I provided evidence that advantageous amino acid substitutions unrelated to IQ repeats have been fixed by adaptive selection in human ASPM after the human-chimpanzee split, which strongly suggests that ASPM might be an important genetic component in the evolutionary expansion of human brain. The episode of positive selection on ASPM appears to have ended some time ago, as there is no evidence for positive selection on ASPM in current human populations; rather, relatively strong purifying selection is detected. Roughly, selective sweeps occurring in the past 0.5N generations may be detected (Fay and Wu 2000), where N is the effective population size of humans and is thought to be ~10,000 (Takahata et al. 1995; Harpending et al. 1998). That is, the positive selection detected in ASPM occurred some time between 6–7 and 0.1 MY ago (0.5 × 10,000 generations × 20 years/generation). The latter date coincides with the suggested time of migration of modern humans out of Africa (reviewed in Cavalli-Sforza and Feldman 2003). It is also interesting to note that although the precise time when positive selection acted on ASPM is difficult to pinpoint, my estimate is consistent with the current understanding that the human brain expansion took place between 2–2.5 and 0.2–0.4 MY ago (McHenry 1994; Wood and Collard 1999). Furthermore, a selective sweep in human FOXP2, a gene involved in speech and language development, has been detected (Enard et al. 2002; Zhang et al. 2002). This sweep was estimated to have occurred no earlier than 0.1–0.2 MY ago (Enard et al. 2002; Zhang et al. 2002). That is, the adaptive evolution of FOXP2 postdated that of ASPM, consistent with the common belief that a big brain may be a prerequisite for language (Decan 1992).

Studies of ASPM in model organisms can help us understand how it impacts brain size. The mouse Aspm is highly expressed in the embryonic brain, particularly during cerebral cortical neurogenesis (Bond et al. 2002). The fruit fly ortholog asp is involved in organizing and binding together microtubules at the spindle poles and in forming the central mitotic spindle (Gonzalez et al. 1990; Wakefield et al. 2001). Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development (Wakefield et al. 2001). The amino acid substitutions in human ASPM are located in exons 3, 18, 20, 21, and 22 (Figure 1), which encode a putative microtubule-binding domain and an IQ calmodulin-binding domain (Bond et al. 2002). These features suggest that the adaptive substitutions in human ASPM might be related to the regulation of mitosis in the nervous system, which can be tested in the future by functional assays of human ASPM as well as a laboratory-reconstructed ASPM protein of the common ancestor of humans and chimpanzees.

Wang & Su, 2004 wrote:Microcephalin gene is one of the major players in regulating human brain development. It was reported that truncated mutations in this gene can cause primary microcephaly in humans with a brain size comparable with that of early hominids. We studied the molecular evolution of microcephalin by sequencing the coding region of microcephalin gene in humans and 12 representative non-human primate species covering great apes, lesser apes, Old World monkeys and New World monkeys. Our results showed that microcephalin is highly polymorphic in human populations. We observed 22 substitutions in the coding region of microcephalin gene in human populations, with 15 of them causing amino acid changes. The neutrality tests and phylogenetic analysis indicated that the rich sequence variations of microcephalin in humans are likely caused by the combination of recent population expansion and Darwinian positive selection. The synonymous/non-synonymous analyses in primates revealed positive selection on microcephalin during the origin of the last common ancestor of humans and great apes, which coincides with the drastic brain enlargement from lesser apes to great apes. The codon-based neutrality test also indicated the signal of positive selection on five individual amino acid sites of microcephalin, which may contribute to brain enlargement during primate evolution and human origin.

Enard et al ,2002 wrote:Language is a uniquely human trait likely to have been a prerequisite for the development of human culture. The ability to develop articulate speech relies on capabilities, such as fine control of the larynx and mouth1, that are absent in chimpanzees and other great apes. FOXP2 is the first gene relevant to the human ability to develop language2. A point mutation in FOXP2 co-segregates with a disorder in a family in which half of the members have severe articulation difficulties accompanied by linguistic and grammatical impairment3. This gene is disrupted by translocation in an unrelated individual who has a similar disorder. Thus, two functional copies of FOXP2 seem to be required for acquisition of normal spoken language. We sequenced the complementary DNAs that encode the FOXP2 protein in the chimpanzee, gorilla, orang-utan, rhesus macaque and mouse, and compared them with the human cDNA. We also investigated intraspecific variation of the human FOXP2 gene. Here we show that human FOXP2 contains changes in aminoacid coding and a pattern of nucleotide polymorphism, which strongly suggest that this gene has been the target of selection during recent human evolution.

Enard et al, 2002 wrote:FOXP2 (forkhead box P2) is located on human chromosome 7q31, and its major splice form encodes a protein of 715 amino acids belonging to the forkhead class of transcription factors2. It contains a glutamine-rich region consisting of two adjacent polyglutamine tracts, encoded by mixtures of CAG and CAA repeats. Such repeats are known to have elevated mutation rates. In the case of FOXP2, the lengths of the polyglutamine stretches differed for all taxa studied. Variation in the second polyglutamine tract has been observed in a small family affected with speech and language impairment, but this did not co-segregate with disorder, suggesting that minor changes in length may not significantly alter the function of the protein4. If the polyglutamine stretches are disregarded, the human FOXP2 protein differs at only three amino-acid positions from its orthologue in the mouse (Fig. 1). When compared with a collection of 1,880 human–rodent gene pairs5, FOXP2 is among the 5% most-conserved proteins. The chimpanzee, gorilla and rhesus macaque FOXP2 proteins are all identical to each other and carry only one difference from the mouse and two differences from the human protein, whereas the orang-utan carries two differences from the mouse and three from humans (Fig. 1). Thus, although the FOXP2 protein is highly conserved, two of the three amino-acid differences between humans and mice occurred on the human lineage after the separation from the common ancestor with the chimpanzee. These two amino-acid differences are both found in exon 7 of the FOXP2 gene and are a threonine-to-asparagine and an asparagine-to-serine change at positions 303 and 325, respectively. Figure 2 shows the amino-acid changes, as well as the silent changes, mapped to a phylogeny of the relevant primates.

We compared the FOXP2 protein structures predicted by a variety of methods6 for humans, chimpanzees, orang-utans and mice. Whereas the chimpanzee and mouse structures were essentially identical and the orang-utan showed only a minor change in secondary structure, the human-specific change at position 325 creates a potential target site for phosphorylation by protein kinase C together with a minor change in predicted secondary structure. Several studies have shown that phosphorylation of forkhead transcription factors can be an important mechanism mediating transcriptional regulation7,8. Thus, although the FOXP2 protein is extremely conserved among mammals, it acquired two amino-acid changes on the human lineage, at least one of which may have functional consequences. This is an intriguing finding, because FOXP2 is the first gene known to be involved in the development of speech and language.