HUMAN MUTATION 28(2), 99^130, 2007

REVIEW ARTICLE

Understanding the Recent Evolution

of the Human Genome: Insights from
HumanChimpanzee Genome Comparisons
Hildegard Kehrer-Sawatzki1 and David N. Cooper2
1
Department of Human Genetics, University of Ulm, Ulm, Germany; 2Institute of Medical Genetics, Cardiff University, Heath Park,
Cardiff, United Kingdom

Communicated by Peter Oefner

The sequencing of the chimpanzee genome and the comparison with its human counterpart have begun to
reveal the spectrum of genetic changes that has accompanied human evolution. In addition to gross karyotypic
rearrangements such as the fusion that formed human chromosome 2 and the human-specific pericentric
inversions of chromosomes 1 and 18, there is considerable submicroscopic structural variation involving
deletions, duplications, and inversions. Lineage-specific segmental duplications, detected by array comparative
genomic hybridization and direct sequence comparison, have made a very significant contribution to this
structural divergence, which is at least three-fold greater than that due to nucleotide substitutions. Since
structural genomic changes may have given rise to irreversible functional differences between the diverging
species, their detailed analysis could help to identify the biological processes that have accompanied speciation.
To this end, interspecies comparisons have revealed numerous human-specific gains and losses of genes as well
as changes in gene expression. The very considerable structural diversity (polymorphism) evident within both
lineages has, however, hampered the analysis of the structural divergence between the human and chimpanzee
genomes. The concomitant evaluation of genetic divergence and diversity at the nucleotide level has
nevertheless served to identify many genes that have evolved under positive selection and may thus have
been involved in the development of human lineage-specific traits. Genes that display signs of weak negative
selection have also been identified and could represent candidate loci for complex genomic disorders. Here,
we review recent progress in comparing the human and chimpanzee genomes and discuss how the differences
detected have improved our understanding of the evolution of the human genome. Hum Mutat 28(2), 99130,
2007. r 2006 Wiley-Liss, Inc.

KEY WORDS: human genome evolution; chimpanzee vs. human genome comparisons; sequence and expression divergence

INTRODUCTION references therein]. That the speciation process of humans and

Irrespective of our anthropocentric viewpoint, hominid evolu-
tion and human speciation are among the most fascinating topics
in evolutionary biology. As opined by McConkey and Goodman
[1997]: Comparative analysis of human and ape genomes is far
more than an excursion into natural history at the molecular level.
Until we have a detailed understanding of the genetic differences
between ourselves and our closest evolutionary relatives, we
cannot really know what we are (p. 351).
The phenotypic divergence evident between humans and
chimpanzees is assumed to have been strongly influenced by
lineage- and species-specific genomic changes that have given rise
to differences in gene expression as well as gains, losses, and
changes in protein function [King and Wilson, 1975; reviewed in
Carroll, 2003; Olson and Varki, 2003; Enard and Paabo, 2004;
Carroll, 2005; Goodman et al., 2005; Varki and Altheide, 2005].
Comparative analyses of the genomes and transcriptomes of
humans and chimpanzees are therefore proving extremely useful in
our attempt to build a picture of the events that have shaped our
respective genomes after their evolutionary divergence, estimated
to have occurred between 5 and 7 million years ago (Mya) [Leakey
et al., 1998; Glazko and Nei, 2003; Kumar et al., 2005; and
chimpanzees may have been rather complex, has been suggested
by DNA sequence divergence determinations using a large
genomic dataset (comprising about 87 Mb) in five primate
species including human and chimpanzee [Patterson et al.,
2006]. It was inferred that the process of human-chimpanzee
speciation began o6.3 Mya. However, after the initial divergence
of the two lineages, further interlineage hybridizations appear to
have occurred prior to the permanent separation of both species
[Osada and Wu, 2005; Patterson et al., 2006].
The Supplementary Material referred to in this article can be
accessed at http://www.interscience.wiley.com/jpages/1059 -7794/
suppmat.
Received 4 May 2006; accepted revised manuscript 10 August
2006.
Correspondence to: Dr. Hildegard Kehrer-Sawatzki, Department
of Human Genetics, University of Ulm, Albert-Einstein-Allee 11,
89081 Ulm, Germany. E-mail: hildegard.kehrer-sawatzki@uni-ulm.de
DOI 10.1002/humu.20420
Published online 5 October 2006 in Wiley InterScience (www.
interscience.wiley.com).

r 2006 WILEY-LISS, INC.

100 HUMAN MUTATION 28(2), 99^130, 2007
The availability of the initial sequence of the chimpanzee
genome has allowed comparison with its human counterpart,
thereby revealing differences and similarities as well as common
patterns of evolution (The Chimpanzee Sequencing and Analysis
Consortium [CSAC]) [Mikkelsen et al., 2005]. The chimpanzee
genome sequence was derived from a single individual of the
Pan troglodytes verus subspecies and represents a draft version
covering
94% of the genome sequence; however, several
segments are not yet anchored within large contigs. This
notwithstanding, the accuracy of the nucleotide sequence is high:
thus, 98% of the assembled sequence has an estimated error rate of
less than 104. This draft sequence therefore provides the means
to analyze both structural and functional aspects of human/simian
genome evolution and the underlying mutational processes
that have molded it. The completion of the chimpanzee genome
sequence and the availability of high quality sequence data
promises not only to help to refine these analyses by excluding
many sequencing errors [Taudien et al., 2006] but should also
potentiate the comparison of highly complex genomic regions such
as the pericentromeric and subtelomeric regions.
The main focus of most human/chimpanzee genome compar-
isons has been the search for lineage-specific differences. Other
primate species have frequently been included in these analyses as
outgroups in order to determine whether the detected differences
occurred in the human or the chimpanzee lineage. As emphasized
by Goodman et al. [2005], comparative sequence data from
other primate species are an invaluable aid to the interpretation of
results obtained from human/chimpanzee genome comparisons,
particularly with respect to the determination of the lineage
specificity of observed differences. The identification of the
genomic differences between humans and chimpanzees, together
with the discovery of the molecular signatures of deviations from
neutral evolution, promise to reveal those proteins and the nature
of the functional networks that have evolved under lineage-
specific adaptive constraints.
We attempt here to summarize recent findings, derived from the
direct comparison of the human and chimpanzee genomes and
transcriptomes, that shed new light on the evolution of the human
genome (Table 1). We explore how human/chimpanzee genome
comparisons (as well as comparative studies involving the other
great apes and Old World monkeys) have contributed to our
understanding of structural (interspecies) divergence and (in-
traspecies) diversity. These studies are likely to have a very
considerable impact not only on our understanding of primate
evolution but also on studies of human disease susceptibility/
complex trait development.
NUCLEOTIDE SEQUENCE DIVERGENCE
Genomewide nucleotide sequence data have allowed the
comparison of 2,400 megabases (Mb) of aligned DNA sequence
between humans and chimpanzees and have confirmed initial
assessments of a rather low overall degree of divergence (inter-
species differences) amounting to only about 1.23% [Mikkelsen
et al., 2005]. Similar rates of divergence have been observed in
previous studies performed with more limited sequence datasets
[Kumar and Hedges, 1998; Chen and Li, 2001; Ebersberger
et al., 2002; Fujiyama et al., 2002; Wildman et al., 2003; Elango
et al., 2006].
For closely related species such as humans and chimpanzees, the
level of nucleotide sequence divergence is strongly influenced
by the degree of polymorphism within the species. Thus, the
observed number of nucleotide differences has to be corrected for
Human Mutation DOI 10.1002/humu
intraspecies polymorphism, which has been estimated to account
for some 11 to 22% of the observed divergence between humans
and chimpanzees [Chen and Li, 2001; Ebersberger et al., 2002;
Elango et al., 2006]. If this polymorphic variation is subtracted
from the overall divergence, the rate of fixed (nonpolymorphic)
sequence divergence between aligned human and chimpanzee
DNA sequence becomes even lower (
1%).
The extent of DNA sequence variation in chimpanzees is some
three-fold higher than that in humans, as determined by the
analysis of a 10-kb region on the X chromosome [Kaessmann
et al., 2001], and
1.5-fold higher according to estimates of
variation across more extensive regions of human, bonobo,
and chimpanzee (eastern, western, and central African) genomes
[Yu et al., 2003]. The lower sequence diversity in humans is
probably due to a combination of the historically lower effective
population size and the recent demographic expansion in the
human lineage.
Humans have a longer generation time than hominoids
(gibbons, orangutan, gorilla, chimpanzees). According to the
generation-time effect hypothesis [Goodman, 1962; Li et al.,
1996], most germline mutations originate from errors in DNA
replication. The longer the generation time, the lower the number
of germ cell replications per unit time, which is in turn reflected in
a smaller number of fixed substitutions. Thus, species with longer
generation times are expected to exhibit slower rates of molecular
evolution than those with shorter generation times. Despite the
relatively low degree of sequence divergence between humans and
chimpanzees, humans exhibit a significant slowdown of molecular
evolution as determined by studies of smaller genomic regions
(e.g. [Li et al., 1987; Bailey et al., 1991; Jaruzelska et al., 1999;
Kaessmann et al., 1999; Shi et al., 2003a; Ebersberger and Meyer,
2005]) and confirmed genomewide employing baboons or rhesus
macaques as outgroups [Elango et al., 2006]. The apparently high
degree of nucleotide sequence conservation manifested by the
chimpanzee and human genomes, a level of conservation which is
particularly pronounced in protein coding regions, is consistent
with the view that the relatively low level of protein divergence
is on its own insufficient to account for the anatomical and
behavioral differences evident between humans and chimpanzees.
Thus, it has been proposed that regulatory changes leading to gene
expression differences are likely to have strongly influenced human
lineage-specific evolution [King and Wilson, 1975; Carroll, 2005].
The extensive nucleotide sequence similarity evident between
the human and chimpanzee genomes simply represents an overall
average value that conceals important local differences. Certain
gene regions manifest considerably more divergence than others
(summarized in Supplementary Table S1; available online at http://
www.interscience.wiley.com/jpages/1059-7794/suppmat). The low-
est extent of sequence divergence is observed in coding regions, a
finding that reflects negative selection against deleterious muta-
tions. 30 untranslated regions (UTRs) have diverged at twice the
rate of coding sequences (Supplementary Table S1). This difference
would appear to be unrelated to the number of hypermutable
CpG sites [Shen et al., 1994a] since 30 UTRs contain on average
about one-half as many CpG sites as coding regions [Hellmann
et al., 2003a]. Obviously, different functional constraints have
shaped the evolution of particular genic regions. The degree of
divergence evident in the 50 UTR is even higher than in the 30 UTR
(Supplementary Table S1). Taking intraspecies diversity into
account, the observed divergence between humans and chimpan-
zees in 50 UTRs is higher than expected and could underlie some
of the gene expression differences noted between humans and
chimpanzees [Enard et al., 2002; Caceres et al., 2003; Karaman

Category
Nucleotide divergence
Recombination pattern
divergence
Divergence/evolution of
proteins
Evolution of theY
chromosome
Human lineage-specic gene
inactivation
Divergence in cancer-related
genes
Expression divergence
Evolution of transcriptional
cis-regulation
Chromosomal dierences
Insertions/deletions
Microsatellite/RY-tract/
triplet repeat evolution
Submicroscopic structural
divergence/segmental
duplications
Human-specic genes/
pseudogenes
Human Mutation DOI 10.1002/humu
HSA, Homo sapiens; PTR, Pan troglodytes.
TABLE 1. Summary of the Main Findings of the Genomewide Human vs. Chimpanzee Genome Comparisons
Results
Number of xed nucleotide substitutions between PTR and HSA:1%.
Regional variation in divergence; substitution rates in subtelomeric regions are elevated in hominids as compared to murids.
Higher divergence in 5and 3 UTRs than in protein coding regions of genes.
Substitution rate at silent sites in exons is lower than at intronic sites, suggesting weak purifying selection operating on silent sites.
Chromosomal variation with highest sequence divergence found on theYchromosome (1.74%) and lowest on the X chromosome (0.94%).
Recombination hotspots appear not be conserved between PTR and HSA.
Genomewide correlation between sequence divergence and recombination rates.
70^80% of proteins are nonidentical between PTR and HSA, although they dier on average by only two amino acid residues.
Average KA/KS ratio is 0.23; the proportion of advantageous mutations in the human lineage is lower than previously estimated but twice that estimated from
the mouse-rat comparison (0.13).
5% of proteins evolved under positive selection specically in the human lineage.
Proteins that have experienced adaptive evolution have been identied in many functional categories.Those associated with brain function/ development may
have been intimately involved in the evolution of human-specic traits.
The absence of any loss or decay of X degenerate genes in HSA as compared to PTR does not favor theimpending demise hypothesis
for the humanYchromosome.
Gene loss or inactivation has not been a rare event during human evolution after the separation from PTR; 90 examples so far known.
Adaptive inactivation which has not yet been completely xed in the human population has been demonstrated in only one case (CASP12 gene)
High degree of overall conservation but a total of 1,542 amino acid changes were identied that could contribute to dierential cancer susceptibility
in PTR and HSA.
The highest degree of expression divergence has been observed in the testis, while the lowest expression divergence has been observed in the brain.
The majority of genes with expression divergence in the brain exhibit a human specic upregulation (increased expression).This human specic upregulation
has not been observed in the liver.
Parallel patterns of gene expression dierences and protein divergence have been detected, suggesting that both types of changes have evolved in concert.
Genes expressed in testis, and especially those located on the X chromosome, display high expression and sequence divergence but reduced diversity in humans,
indicating that genes involved in reproduction evolved under positive selection.
Adaptive evolution of human upstream regulatory regions l has been proven for the prodynorphin (PDYN) and the factorVII (F7) genes.
Nine cytogenetically detectable pericentric inversions and one fusion that gave rise to human chromosome 2 serve to distinguish PTR and HSA; no genes were
disrupted at the breakpoints.Two inversions (chromosomes 1 and 18) were xed in the human lineage.
Insertions of 10 to 15 kb have given rise to a total of
32 Mb of human-specic DNA sequence.
Insertions of 415 kb gave rise to
8 Mb of human-specic sequences.
Genomewide comparisons have indicated that some 40^45 Mb of lineage-specic sequence results from insertions/deletions resulting in copy number
dierences between HSA and PTR.
The number of Alu sequence insertions in HSA was
3.4-fold higher than in PTR. A dierent set of Alu sequences has been amplied in both species, while the
rate of L1 element retrotranspositional activity has been relatively similar in HSA and PTR.
Direct genome comparisons revealed
1,000 SVA retrotransposons specic to the human lineage and a comparable number of chimpanzee
lineage-specic integrations.
The large number of lineage-specic transposon insertions in PTR and HSA could have had functional consequences with respect
to gene expression or inactivation.
45 chimpanzee-specic endogenous retroviral element insertions and 73 human-specic insertions have been identied.
Microsatellites, triplet-repeats, and RY-tracts tend to be longer in HSA than in PTR suggesting that expansion-type mutations have occurred disproportionately
in the human lineage.
33% of segmental duplications (26.5 Mb) are duplicated only in HSA; they map to at least 500 distinct regions of average length
55 kb and are distributed
throughout the genome.
There are many structural dierences in subtelomeric regions of HSA compared to PTR.
Microinversions of 41kb up to several Mb contribute signicantly to genome divergence and (intraspecies) diversity.
At least 180 genes and partial gene sequences have become duplicated specically in the human genome.
HUMAN MUTATION 28(2), 99^130, 2007
Humans manifest a larger number of lineage-specic gene duplications than the great apes;134 gene families have been identied with human lineage-specic
gains including a number of genes with brain-related functions.
163 human-specic retrotransposed gene copies have been identied.
101
102 HUMAN MUTATION 28(2), 99^130, 2007
et al., 2003; Marvanova et al., 2003; Uddin et al., 2004;
Khaitovich et al., 2005a].
The nucleotide substitution rate in nondegenerate sites within
coding sequences is substantially lower than expected, probably a
reflection of the action of purifying selection. Similarly, divergence
at silent sites (in particular four-fold degenerate sites where
substitutions do not affect the encoded protein) is often lower
than at nearby intronic locations or within intergenic regions, a
finding that is also consistent with the action of purifying selection
[Hellmann et al., 2003a; Mikkelsen et al., 2005]. Even if CpG
sites are disregarded, the divergence at four-fold degenerate sites is
39% lower than in intergenic regions [Hellmann et al., 2003a].
This is in accord with the study of Bustamante et al. [2002], who
compared synonymous sites between functional human genes
and pseudogenes. The reasons why purifying selection may have
acted on four-fold degenerate sites in humans may include codon
usage bias [Lander et al., 2001], isoacceptor tRNA preference
[Sharp and Li, 1986] and an influence on mRNA splicing. In
humans, codon usage bias is positively correlated with the breadth
of the genes tissue expression profile [Urrutia and Hurst, 2001],
conservation of mRNA secondary structure, and regulation of
mRNA splicing, as discussed by Hellmann et al. [2003a].
Human/chimpanzee genome comparisons have revealed regio-
nal differences in sequence divergence such as dark G-bands
on chromosomes. These bands are both G1C-poor and gene-poor,
exhibit a low recombination frequency [Holmquist, 1992], and
display a degree of divergence that is 10% higher than the
genome average. By contrast, greater divergence is evident
within the gene-rich and G1C rich regions at the ends of those
chromosomes that exhibit high recombination rates [Hellmann
et al., 2003b]. The variable degree of divergence in different
chromosomal regions is probably influenced by a complex interplay
between different parameters such as gene content and recombi-
nation frequency, together with other factors (e.g., variation in the
mutation rate, biased gene conversion, and the DNA sequence
context) [Lercher and Hurst, 2002; Meunier and Duret, 2004].
Remarkably, neither the sequence divergence nor the recombina-
tion rate in distal chromosomal regions is increased in mouse or rat
[Jensen-Seaman et al., 2004], in contrast to the situation in
hominids (humans and great apes). Remarkably, the sequence
divergence in the most distal 10 Mb of hominid chromosomes is

10% higher than in rest of the genome [Mikkelsen et al., 2005].
By contrast, in the nondistal regions of chromosomes, divergence
is strongly correlated between hominids and murids. Accordingly,
a lineage-specific increase in sequence divergence may be noted
in hominids in the gene-rich distal chromosomal regions
[Mikkelsen et al., 2005].
Considerable regional differences in sequence evolution are
evident on the Y chromosome [Ebersberger and Meyer, 2005]. In a
detailed comparison of a 90-kb region from the nonrecombining
portion of the Y chromosome between humans and chimpanzees
(using gorilla as an outgroup), significantly different region-specific
substitution rates have been observed. These differences were
found to be associated with a change in GC content that is
confined to the human lineage (and is not evident in the
chimpanzee). Recent species-specific changes in the regions
mutation pattern (as well as selective effects) may thus have
driven the sequence divergence in this chromosomal region in
both humans and chimpanzees [Ebersberger and Meyer, 2005].
Nucleotide sequence divergence varies not only within but
also between chromosomes [Chen et al., 2001; Ebersberger
et al., 2002; Fujiyama et al., 2002; Smith et al., 2002; Mikkelsen
et al., 2005; Hughes et al., 2005; Kuroki et al., 2006]. The
Human Mutation DOI 10.1002/humu
whole-chromosome comparison of human chromosome 21 and
its chimpanzee homologue has indicated considerably higher
divergence (1.44%) than the overall value for the genome
[Watanabe et al., 2004]. By contrast, the X chromosome exhibits
the lowest sequence divergence for any chromosome (0.94%)
whereas the Y chromosome displays the highest (1.78% divergence
as compared to the overall value of 1.23%) [Hughes et al.,
2005; Kuroki et al., 2006; Patterson et al., 2006]. This is probably
a consequence of the elevated germline mutation rate in males
[Li et al., 2002; Makova and Li, 2002; Mikkelsen et al., 2005;
Taylor et al., 2006].
RECOMBINATION PATTERN DIVERGENCE
BETWEEN HUMANS AND CHIMPANZEES
Comparative analyses of recombination patterns in humans
and chimpanzees may shed light on the evolution of a key
mechanism which underlies the generation of genetic diversity
(reviewed by Nishant and Rao [2006]). Comparison of linkage
disequilibrium landscapes in humans and chimpanzees has
revealed major divergence in haplotype block structures and
hence, by inference, in the positions of recombination hotspots
[Wall et al., 2003; Ptak et al., 2004, 2005; Winckler et al., 2005].
In view of the low degree of sequence divergence between humans
and chimpanzees, changes in local DNA sequence motifs are
unlikely to be the sole determinants of the differential recombina-
tion hotspot distribution.
Despite differences in the crossover hotspot patterns in humans
and chimpanzees, a common gradient of increasing recombination
rate is apparent in the pseudoautosomal region on the short arm
(p-PAR) of the X chromosome in both species [Filatov, 2004]. The
mutagenic influence of recombination may make a significant
contribution to the gradual increase in silent substitution rates
that extends from the proximal to the distal portion of the p-PAR.
Furthermore, the between-species silent divergence in the
p-PAR also forms a gradient that increases towards the telomere
[Filatov, 2004; Bussell et al., 2006]. The correlation between
human-chimpanzee sequence divergence and recombination
rates is not restricted to the p-PAR but rather is observed
genomewide, suggesting that mutation and recombination
are intimately associated processes [Lercher and Hurst, 2002;
Hellmann et al., 2003b].
TRANSSPECIES OR SHARED POLYMORPHISMS
At the nucleotide sequence level, shared polymorphisms among
higher primate species are likely to be rare. They do however exist,
as is to be expected in cases of independently occurring mutations
or where positive selection for diversity has had a major influence
on allele frequencies, as with the human leucocyte antigen (HLA)
genes [Watkins, 1995]. Only a few shared human/chimpanzee
RFLPs [Mountain and Cavalli-Sforza, 1994] or SNPs [Martinko
et al., 1993; Deeb et al., 1994; Jaeger et al., 1998; Hacia et al.,
1999] have been identified to date. This may be in part because,
with the sole exception of the study of Asthana et al. [2005], no
systematic attempt has yet been made to search for shared
polymorphisms between humans and chimpanzees.
Consistent both with theoretical expectations [Kimura, 1983]
and empirical observations [Kennedy et al., 2003], the vast
majority of identified human SNPs have had a relatively recent
evolutionary origin and are not shared by other primate species.
Thus, the probability that a polymorphism originally present
in the common ancestor of chimpanzees and humans would have

survived for 4.6 million years (Myrs) appears to be on the order of

2  106 [Asthana et al., 2005]. It therefore follows that most
polymorphisms shared by humans and chimpanzees are more likely
to be the result of independent mutational events in both lineages
rather than being truly identical-by-descent. This notwithstand-
ing, the probable extreme rarity of shared identical-by-descent
polymorphisms renders their occasional identification all the
more meaningful since they could at least in principle provide
information on relative rates of mutation, recombination, gene
conversion, and even more importantly, evidence of positive
selection due to heterozygote advantage.
Plausible human examples of balanced functional polymorph-
isms fulfilling a role in defense against pathogens are to be found in
the chemokine receptor [Tang et al., 2002], the HLA gene clusters
[Gao et al., 2001], as well as the cytokine network [Shin
et al., 2000]. Although these examples have originated relatively
recently in human evolution, the maintenance of variants in both
species is indicative of rather more ancient selective pressures. The
identification of shared identical-by-descent polymorphism in
humans and chimpanzees could serve to pinpoint genomic regions
harboring loci that manifest heterozygote advantage. However,
humanchimpanzee genome comparisons have also proven useful
in excluding the possibility of shared heterozygote advantage, as in
the case of the variants that impart bitter taste sensitivity;
variation at the TAS2R38 locus, the molecular basis of taste
sensitivity, has been shown to have arisen independently in the
two primate species [Wooding et al., 2006].
ADAPTIVE EVOLUTION OF PROTEINS
Differences in the evolutionary rates of genes indicate that both
positive and negative selection have operated extensively on the
human and chimpanzee genomes [Lu and Wu, 2005]. Genes that
have experienced positive selection specifically in the human
lineage, promise by virtue of their function to identify the
molecular mechanisms responsible for the development of human
specializations [Sabeti et al., 2006].
An estimated 70 to 80% of protein sequences are nonidentical
between humans and chimpanzees, although these proteins differ
by on average only two amino acid residues [Glazko et al., 2005;
Mikkelsen et al., 2005]. To establish whether the fixation of
amino acid replacements has been a consequence of positive
selection (and hence may be regarded as adaptive changes), the
KA/KS ratios (representing the relative ratio of nonsynonymous/
synonymous substitutions) are determined as pairwise values
between the human and chimpanzee sequences [Kimura, 1968;
Eyre-Walker and Keightley, 1999]. If KA/KS41, then the gene in
question may be considered to have evolved under directional
positive selection [Yang and Bielawski, 2000]. Neutrally
evolving genes generally have KA/KS values around 1, whereas
most proteins have KA/KS values r1 and hence may be
considered to be under purifying or negative selection. The power
to detect genes that have evolved under positive selection by KA/
KS determination using the complete coding sequence is, however,
often quite low [Kitano et al., 2004; Ellegren, 2005; Nielsen et al.,
2005] and even genes with KA/KS values o1 may have evolved
under positive selection [Dorus et al., 2004]. Furthermore, the
significance of single adaptive amino acid changes within
crucial functional domains may not be immediately apparent
[Andres et al., 2004a], particularly if negative selection has
acted upon other sites in the protein in question and the overall
KA/KS value is less than 1.
HUMAN MUTATION 28(2), 99^130, 2007 103
In order to factor some of the above considerations into tests for
deviation from neutrality based on KA/KS ratios, different methods
have been developed to search for positive selection acting on
given branches or on a subset of sites (summarized in Zhang
et al. [2005a]). The use of different methods may explain certain
discrepancies between studies designed to identify genes under
positive selection in the human lineage [Clark et al., 2003; Dorus
et al., 2004; Mikkelsen et al., 2005; Nielsen et al., 2005; Arbiza
et al., 2006]. In a set of 13,454 human/chimpanzee orthologous
gene pairs, the CSAC identified 585 genes exhibiting KA/KS ratios
41 in the human lineage as being potential candidates for positive
selection [Mikkelsen et al., 2005]. This proportion was lower
than estimates obtained from two other genomewide screens
for positively selected genes in human/chimpanzee comparisons
[Clark et al., 2003; Nielsen et al., 2005]. In addition, only a subset
of genes was identified as having evolved under positive selection
common to all three studies.
More recently, Arbiza et al. [2006] employed a two branch-site
maximum likelihood (ML) method corrected for multiple testing
on a set of 13,198 genes with human, chimpanzee, mouse, rat,
and dog orthologs. According to their findings, some 5% of
human genes and 10% of chimpanzee genes have evolved under
positive selection. The higher number of positively selected
genes in chimpanzees as compared to humans may have been a
consequence of the smaller effective population sizes during
human evolution [Chen and Li, 2001].
Arbiza et al. [2006] also observed that many functional
categories were overrepresented among the genes found to have
evolved under positive selection, for example nociception/sensory
perception, brain-related functions and development, pathogen
resistance, immunity, reproduction, dietary adaptation, and trans-
criptional regulation. These observations are in accord with the
results of Clark et al. [2003] as well as findings from studies that
employed more limited datasets and those that focused upon
individual genes [Yang, 1998; Wyckoff et al., 2000; Enard et al.,
2002; Swanson and Vacquier, 2002; Choi and Lahn, 2003; Gilad
et al., 2003a; Maier et al., 2003; Wang et al., 2003; Bersaglieri
et al., 2004; Dorus et al., 2004; Kitano et al., 2004; reviewed in
Vallender and Lahn, 2004a; Bustamante et al., 2005; Clark and
Swanson, 2005; Nielsen et al., 2005; Dorus et al., 2006; Verrelli
et al., 2006; Voight et al., 2006]. Several of the above-mentioned
biological processes, in particular pathogen resistance and
reproduction, are likely to have been influenced by selection not
only in humans but also in chimpanzees [Arbiza et al., 2006]. By
contrast, adaptive evolution associated with brain function/
development may have been intimately related to the emergence
of human-specific traits.
Of especial interest are those genes that have not only evolved
under positive selection in early primate evolution but which
also still display signatures of positive selection postdating
the emergence of anatomically modern humans, e.g., micro-
cephalin (MCPH1) and abnormal spindle-like microcephaly-associated
(ASPM). Both these genes may influence brain size since muta-
tions in them cause primary microcephaly, a neurodevelopmental
disorder characterized by a global reduction in cerebral cortical
volume but with no major abnormalities in cortical architecture
[Bond et al., 2002; Jackson et al., 2002; Roberts et al., 2002].
Remarkably, both genes display evidence of strong selection in the
primate lineage leading to humans. The ASPM gene experienced
pronounced positive selection in the African hominoid clade
[Evans et al., 2004a; Kouprina et al., 2004a], whereas the MCPH1
gene did so in an earlier phase of primate evolution. Strong
positive selection has impacted upon the MCPH1 gene in New
Human Mutation DOI 10.1002/humu
104 HUMAN MUTATION 28(2), 99^130, 2007
World monkeys, Old World monkeys, gibbons, and orangutan
[Evans et al., 2004b]. Intriguingly, both brain-expressed genes
appear to have been continuing their trend of adaptive evolution
since certain variants or haplotypes have been reported to increase
in frequency in human populations more rapidly than would be
predicted by random drift alone [Evans et al., 2005; Mekel-Bobrov
et al., 2005]. The biological basis for this selection (for example,
whether it is associated with the proliferation of neural progenitor
cells) is unclear, but its elucidation promises to yield new
insights into basic mechanisms of human brain evolution [Stern
and Woods, 2006].
Interestingly, MCPH1 is located in a very rapidly evolving
15-Mb region on chromosome 8p; this region displays an extremely
high degree of nucleotide sequence divergence between human
and chimpanzee, the highest so far reported for the autosomes
[Nusbaum et al., 2006]. Apart from the MCPH1 gene, this region
also contains other genes that appear to have evolved under
positive selection in primates, such as the major defensin gene
cluster [Boniotto et al., 2003; Maxwell et al., 2003; Semple et al.,
2003, 2005; Xiao et al., 2004]. The defensin genes encode a family
of peptides that are involved in the immune response [Lehrer,
2004] and may play a role in conferring differential disease
susceptibility in humans. Genes and segmental duplications within
the defensin gene cluster exhibit considerable copy number
variation between human populations [Mars et al., 1995; Hollox
et al., 2003; Aldred et al., 2005] as well as haplotype diversity
[Taudien et al., 2004], suggesting persistent adaptive evolution in
humans and chimpanzees. It is possible that MCPH1 variants
could have become fixed by hitchhiking along with new defensin Some of these genomic regions, such as that on chromosome 22,
variants as they themselves became fixed.
The adaptive evolution of brain-expressed genes in primates is
unlikely to be restricted to ASPM and MCPH1. Indeed, 17 out of
24 genes with marked KA/KS disparities between primates and
rodents are linked to brain development and physiology [Dorus
et al., 2004]. However, the issue of lineage specificity remains
questionable, since the accelerated evolution of these genes
has also been observed in the dog lineage as compared to mouse
[Lindblad-Toh et al., 2005]. When human and chimpanzee are
compared, genes with maximal expression in the brain exhibited
little or no evidence of positive selection in the human lineage
[Nielsen et al., 2005]. Further, of 2,633 human brain-expressed
genes, only 47 displayed evidence of positive selection in the
human lineage but not in chimpanzees [Yu et al., 2006].
These findings support the view that cognitive differences might
be related to adaptive changes in only a few genes with low
or regionally restricted expression in the brain and/or changes in
gene expression.
The insufficient sensitivity of whole-genome analyses based on
relative ratio tests to detect all genes under positive selection is
evidenced by examples such as FOXP2, which was not identified
as being positively selected by Nielsen et al. [2005] and Arbiza
et al. [2006]. FOXP2 is an attractive candidate for involvement in
human speech development. Carriers of FOXP2 mutations
display impairment in writing, verbal articulation (developmental
verbal dyspraxia [DVD]), and grammatical usage [Lai et al., 2001;
Bishop, 2002]. Although the FOXP2 gene has been very highly
conserved in mammals, two amino acid changes occurred
specifically in the human lineage; this was accompanied by the
loss of local DNA sequence diversity in the FOXP2 gene region
[Enard et al., 2002; Zhang et al., 2002a]. Such a selective sweep those amino acid substitutions that have occurred are likely to be
generally occurs when an advantageous allele arises and spreads
rapidly through the population. Concomitantly, adjacent regions
hitchhike with the advantageous site and display a genealogy that
Human Mutation DOI 10.1002/humu
is very different from that expected in the absence of selection
[Maynard Smith and Haigh, 1974; Fay and Wu, 2000; Bamshad
and Wooding, 2003; Enard and Paabo, 2004]. The selective sweep
in the FOXP2 region appears to have occurred during the last
100,000 years [Zhang et al., 2002b], a time period that overlaps
the emergence of modern humans and the acquisition of their
language capability. Although FOXP2 may indeed be causatively
associated with this most characteristic of human features, it is
not necessarily going to be unique in this regard. Moreover, as a
transcription factor whose activity is not restricted to the brain,
FOXP2 clearly does not control language capability directly but
rather may either play a more indirect role in a neuronal pathway
that regulates language acquisition or instead regulate anatomical
requirements for speech development outside the brain. Human
language ought not to be viewed as an innovation that occurred
specifically (and in its entirety) in the human lineage but rather
perhaps as a complex reconfiguration of preexisting systems that
evolved coordinately (reviewed by Fisher and Marcus [2006]).
Thus, the identification of genes that evolved under recent
positive selection may help us to identify the mechanistic origins of
human speech development.
Regions in the human genome where diversity is lower than
expected (as deduced from the divergence rate with the
chimpanzee) can help to pinpoint regions that have evolved
under positive selection in the human lineage. Mikkelsen et al.
[2005] identified six such regions of reduced diversity that serve as
indicators of strong selective sweeps that have acted during the
last 250,000 years of human evolution (Supplementary Fig. S1).
are quite gene-rich, whereas others, such as that on chromosome
4, do not contain any genes. Detailed analyses of the genetic
variation patterns of these regions can help to identify genes or
regulatory elements that have been the target of selection.
Some major differences between humans and chimpanzees
with respect to disease (in particular, cancer) susceptibility have
been noted (summarized by Varki and Altheide [2005]). Cancer-
related genes may have experienced greater selective pressure than
other disease genes [Thomas et al., 2003]; for example, adaptive
evolution of the breast cancer (BRCA1) and angiogenin (ANG)
genes has been evident during primate evolution [Huttley et al.,
2000; Zhang and Rosenberg, 2002; Pavlicek et al., 2004]. Puente
et al. [2006] identified a total of 1,542 amino acid changes in 333
cancer-related genes that could, at least in principle, contribute to
a possible differential cancer susceptibility between humans and
chimpanzees. In addition, 20 cancer-related genes were identified
that contain codon insertions or deletions in their protein
coding regions when humans and chimpanzees are compared;
70% of these occurred within trinucleotide repeats, underlining
the genetic instability of these sequences (summarized in
Supplementary Table S2).
NEGATIVE SELECTION
IN THE HUMAN LINEAGE
Genes with signatures of negative selection (KA/KSr1) are
also potentially important in the context of medical
genetics. Genes that have evolved under negative or balancing
selection attract attention by virtue of the minimal amino acid
sequence divergence between humans and chimpanzees since
deleterious to differing extents and hence are less likely to
become fixed. Such a pattern was evident in 13.5% of
6,000
genes analyzed by Bustamante et al. [2005]. Those human genes

that manifest low sequence divergence but a high degree of
intraspecific diversity are particularly worthy of note since these
genes could harbor an excess of mildly deleterious variants.
Interestingly, cytoskeletal proteins and proteins involved in both
ectoderm development and neurogenesis display signatures of
negative selection with an excess of amino acid polymorphism
relative to divergence. A substantial proportion of genes with
signatures of negative selection are known to be associated with
Mendelian disorders (including Duchenne muscular dystrophy,
Huntington disease and Usher syndrome among others [Busta-
mante et al., 2005]). It follows that weak negative selection
signatures could prove useful in identifying genes associated with
complex disorders.
EVOLUTION OF THE HUMAN Y CHROMOSOME
The Y chromosome has a number of unique features; i.e., it is
haploid and male-specific. Owing to its lower effective population
size as compared to other chromosomes, selection acting on the Y
chromosome is necessarily weaker. On the other hand, the Y
chromosome evolves more rapidly than other chromosomes due to
the higher mutation rate in the male germline [reviewed in Jobling
and Tyler-Smith, 2003; Tyler-Smith et al., 2006]. X and Y
chromosomes coevolved from an ancestral pair of autosomes that
existed
300 Mya [Lahn and Page, 1999]. Since then, the Y
chromosome has degenerated gradually both in terms of gene
content and size, because over most of its length it does not
recombine during meiosis [Charlesworth and Charlesworth, 2000].
The considerable degree of evolutionary decline has suggested to
some that the Y chromosome could become devoid of any
functional gene within 10 Myrs [Aitken and Marshall Graves,
2002]. As a means to investigate this impending demise
hypothesis, which implies that the Y chromosome is continually
losing genes shared with the X, the comparison of Y-linked genes
in humans and chimpanzees has proven very informative. Hughes
et al. [2005] compared 16 Y chromosomal genes and 11
pseudogenes located in the so called X degenerate regions of rodents) is that of the olfactory receptor (OR) genes [Rouquier
humans and chimpanzees. These genes have diverged substantially
during the period of separated evolution of the X and Y
chromosomes and are not involved in recombination or gene
conversion as are genes located in the palindromic sequences that
are abundant on the Y chromosome [Rozen et al., 2003; Skaletsky
et al., 2003]. These comparisons indicated no significant loss or
decay of these genes in the human lineage [Hughes et al., 2005]
and the results of these analyses were confirmed by comparison
with another chimpanzee individual [Kuroki et al., 2006]. This
observation is clearly inconsistent with the impending demise
hypothesis for the human Y chromosome since no gene loss or
decay has occurred during the last
5 to 7 million years of human
evolution. Furthermore, human/chimpanzee sequence compari-
sons revealed pronounced coding sequence conservation in the X
degenerate genes, indicating that their function has been
maintained by purifying selection [Hughes et al., 2005].
COMPARATIVE EVOLUTION OF THE HUMAN
AND CHIMPANZEE MITOCHONDRIAL GENOMES
Comparison of the complete sequences of the human and
chimpanzee mitochondrial genomes has yielded a value of 8.9% for
the nucleotide sequence divergence between the two primate
species [Arnason et al., 1996]. Corrected for the actual mutation
rates, however, divergence estimates have sometimes been
reported to be twice as high. Although the high level of
HUMAN MUTATION 28(2), 99^130, 2007 105
interspecies homoplasy makes genegene direct comparisons
difficult between human and chimpanzee mitochondrial sequences
[Kivisild et al., 2006], mitochondrial genome diversity does appear
to be markedly higher in chimpanzees than in humans, possibly as
a result of recent human population bottlenecks [Wise et al.,
1997].
Sequence transfer from the mitochondrial genome to the
nuclear genome has been an ongoing process during mammalian
evolution. Although the precise mechanism of sequence transfer
is unclear, more than 500 of these nuclear DNA sequences of
mitochondrial origin (NUMTs) have been identified in the human
genome. Further, a total of 27 NUMTs have been identified as
being human-specific, having been inserted into the human
nuclear genome over the last 5 to 7 Myrs [Ricchetti et al., 2004.
HUMAN LINEAGE-SPECIFIC GENE INACTIVATION
The less is more hypothesis has emphasized the importance of
loss-of-function mutations during human genome evolution
[Olson, 1999; Olson and Varki, 2003]. In this context, gene loss
or pseudogenization could have had a much greater impact than
most amino acid replacements due to the immediate loss of gene
function [Wang et al., 2006]. A good example of this principle is
provided by MYH16, a myosin heavy chain gene expressed in the
jaw musculature and inactivated by a human-specific truncating
mutation that is thought to have originated
2.4 Mya [Stedman
et al., 2004]. This inactivation event therefore predates the
emigration of Homo from Africa and could represent a molecular
adaptation that enabled remodeling of the hominid cranium by
reducing masticatory musculature.
Numerous other examples of human-specific gene inactivation
events have been identified (listed in Table 2). These inactivations
were either caused by nonsense/frameshift mutations or by the loss
of paralogous gene copies that were duplicated during hominoid
evolution. One gene family that has been subject to extensive
gene loss in the human and primate lineages (as compared to
et al., 1998; Sharon et al., 1999; Young et al., 2005], one of the
largest mammalian gene superfamilies, comprising 41,000 genes
[Young and Trask, 2002; Grus et al., 2005]. Remarkably, humans
have accumulated mutations that have disrupted OR gene coding
regions roughly four-fold faster than the great apes or macaques.
Of 50 human OR genes comparatively investigated in humans and
other primates, 54% were found to contain inactivating mutations
and thus represent pseudogenes [Gilad et al., 2003b]. Although
the pseudogenization of OR genes has also been noted in
nonhuman primates, a much higher attrition rate in terms of
functional OR genes has occurred in the human lineage, possibly
due to reduced chemosensory dependence as compared to the
great apes [Gilad et al., 2003b].
Another very interesting example of gene inactivation,
occurring specifically in the human lineage, was mediated by an
Alu insertion into the CMP-Neu5Ac hydroxylase (CMAH) gene,
an event that occurred after the separation of humans and
chimpanzees. Owing to this deficiency, N-acetylneuraminic acid
(Neu5Ac) cannot be transformed into N-glycolylneuraminic acid
(Neu5Gc), which is consequently absent in humans but present in
the great apes [Chou et al., 1998; Irie et al., 1998; Muchmore
et al., 1998; Hayakawa et al., 2001]. Neu5Gc and Neu5Ac are the
major sialic acids that function as cell-surface glycoconjugates and
appear to be responsible for the observed humanchimpanzee
differences in susceptibility to malaria [Martin et al., 2005].
Interestingly, loss of Neu5Gc would appear to have initiated other
Human Mutation DOI 10.1002/humu
 TABLE 2. Genes Inactivated in the Human Lineage but Functional in the Chimpanzee
106

Chromosomal
Gene Pseudogene description Functional context (type of inactivating mutation) localization References

BASE Breast cancer and salivary gland Unknown (frameshift 1-bp deletion) 20 Hahn and Lee [2005]; Wang et al. [2006]
expression
DNAJB3 DnaJ (Hsp40) homolog, subfamily B, Heat shock protein binding (frameshift 1-bp insertion) 2 Hahn and Lee [2005]; Wang et al. [2006]
member 3
FLJ33674 Hypothetical protein FLJ33674 Unknown (frameshift deletion) 3 Hahn and Lee [2005]; Wang et al. [2006]
HEJ1 Transcribed processed pseudogene Unknown (insertion of 2 nucleotides) 1 Hahn and Lee [2005]
of DNAJA1
NTSR2 Neurotensin receptor 2 Levocabastine-sensitive G protein-coupled neurotensin 2 Hahn and Lee [2005]

Human Mutation DOI 10.1002/humu

receptor 2 (frameshift deletion)
RPL13AP Transcribed processed pseudogene Unknown (insertion of 2 nucleotides) 14 Hahn and Lee [2005]
of RPL13A
SCGB1D4 Secretoglobin family 1D member 4 Expression is inducible by interferon-g; involved in 11 Hahn and Lee [2005]
chemotactic migration and in invasion of lymphoblast
cells (frameshift deletion)
WBSCR27 Williams Beuren syndrome chromosome Unknown (frameshift 11-bp insertion) 7q11.23 Hahn and Lee [2005]; Wang et al. [2006]
region 27
ZCCHC13 Zinc nger, CCHC domain containing 13 DNA binding (1-bp insertion) X Hahn and Lee [2005]
HUMAN MUTATION 28(2), 99^130, 2007

CML2 Putative N-acetyltransferase Homology to bacterial acetyltransferases involved in drug 2p13.1 Hahn and Lee [2006]
Camello-like 2 resistance (nonsense mutation)
FLJ14640 Hypothetical protein ATPase domain; function unknown (nonsense mutation) 19q13.11 Hahn and Lee [2006]
MT1L Metallothionein 1L Heavy metal binding activity (nonsense mutation) 16q13 Hahn and Lee [2006]
NPPA Natriuretic peptide precursorA Ligand- and retinoid X receptor-dependent, hormonal 1p36.21 Hahn and Lee [2006]
activity (nonsense mutation)
PDE3B Phosphodiesterase 3B, cGMP-inhibited 3 0, 50 -cyclic-nucleotide phosphodiesterase activity; 11p15.1 Hahn and Lee [2006]
hydrolase activity (nonsense mutation)
SERPINA13 Serine peptidase inhibitor, clade A, Serine-type endopeptidase inhibitor activity 14q32.13 Hahn and Lee [2006]
member 13 (nonsense mutation)
TAP2 Transporter 2, ATP-binding cassette, Membrane-associated, involved in multidrug resistance, 6p21.3 Hahn and Lee [2006]
sub-family B heterodimerization withTAP1 (nonsense mutation)
UIP1 26S proteasome-associated UCH Interaction with ubiquitin C-terminal hydrolase Xq28 Hahn and Lee [2006]
interacting protein 1 37 (nonsense mutation)
ZNF277 Zinc nger protein (C2H2 type) 277 Zinc ion binding, transcription factor activity (nonsense 7q31.1 Hahn and Lee [2006]
mutation)
MYH16 Myosin,heavy polypeptide 16 Sarcomeric myosin heavy chain expressed in non-human 7q22.1 Stedman et al. [2004]; Wang et al. [2006]
primate masticatory muscles (frameshift mutation)
CASP12P1 Caspase-12 pseudogene Cysteine protease that cleaves C-terminal 11q22.3 Fischer et al. [2002]; Saleh et al. [2004];
aspartic acid residues on their substrate molecules Wang et al. [2006]
(nonsense mutation)
FMO2 Flavin containing monooxygenase 2 NADPH-dependent enzyme that catalyzes the oxidation of 1q23^q25 Dolphin et al. [1998]
many drugs and xenobiotics (nonsense mutation)
TCRGV10 Tcell receptor gamma variable 10 (point mutation in theV10 leader donor splice site) 7p15 Zhang et al. [1996]
OR 36 olfactory receptor (OR) pseudogenes Interaction with odorant molecules to initiate a neuronal Gilad et al. [2003b]; Wang et al. [2006]
response and perception of smell (nonsense mutations)
ELA1 Pancreatic elastase 1 Serine protease (inactivating substitutions in enhancer and 12q13 Rose and MacDonald [1997]
promoter)
KRTHAP1 Keratin, hair, acidic pseudogene 1 Intermediate lament (nonsense mutation) 17q12^q21 Winter et al. [2001]
TAS2R62P, Bitter taste receptor pseudogenes Bitter taste reception (nonsense mutation) 7q35,7p21.3, Conte et al. [2002]; Shi et al. [2003b];
TAS2R2, 12p13.2 Wang et al. [2004, 2006]; Go et al.
TAS2R64P [2005]
NAPSB Napsin B peptidase pseudogene Napsin B aspartic peptidase (nonsense mutation) 19q13.33 Puente et al. [2005]
 HUMAN MUTATION 28(2), 99^130, 2007 107

adaptive changes in human sialic acid biology affecting Siglec-9

Hamann et al. [2003]; Wang et al. [2006]

and Siglec L1 (Siglec XII), sialic acid-binding receptors of innate

Muchmore et al. [1998]; Hayakawa

immune cells [Angata et al., 2001; Sonnenburg et al., 2004].

Chou et al. [1998]; Irie et al. [1998];

et al. [2001]; Wang et al. [2006]

Further, the human-specific loss of Siglec expression on T cells is
associated with Tcell hyperactivity, which might itself be related to
the striking differences between humans and chimpanzees in terms
McEvoy and Maeda [1988]

of both the prevalence and severity of T-cell mediated diseases,

such as acquired immunodeficiency syndrome (AIDS) [Nguyen
Angata et al. [2004]

et al., 2006a].
Jury et al. [1998]

In addition to the human-specific gene inactivation events

listed in Table 2, Wang et al. [2006] identified a further 67
pseudogenes that have been generated specifically in the human
lineage after the split from the chimpanzee lineage. This serves
to indicate that gene losses restricted to the human lineage are by
no means rare occurrences. Wang et al. [2006] allocated these
67 pseudogenes, together with 13 previously described human-
12q24.12^q24.13

specific pseudogenes, to functional categories. An overrepresenta-

6p21.32
19p13.3

tion of functional categories such as chemoreception, immune

16q22

response, and calcitonin receptor activity was noted, which might

be associated with species-specific physiological differences in
olfaction [Glusman et al., 2001; Shepherd, 2004] or pathogen
susceptibility [Escalante et al., 1995; Novembre et al., 1997]. The
genes involved in the latter two processes are known to have
Metallopeptidase domain 1 (small insertions, deletions and
Present in hominoids, but deleted in humans. Hominoids

evolved rapidly [Grus et al., 2005; Kelley et al., 2005] but the
G-protein coupled receptor activity, calcium ion binding,

contribution of adaptive gene loss within these functional

N-glycolylneuraminic acid (Alu repeat insertion)

categories remains to be clarified [Wang et al., 2006].

Immunoglobulin superfamily member lectins that

Although the importance of the aforementioned gene inactiva-

Converts sialic acid N-acetylneuraminic acid to
selectively recognize sialic acids (gene loss)
integral to membrane (nonsense mutation)
have 3 haptoglobin genes, humans have 2

tion events for human evolution remains to be established, the

idea encapsulated in the gene loss hypothesis is highly attractive
since it provides a mechanism whereby trait evolution can occur
rapidly without being dependent upon the occurrence of several
genetic alterations acting additively, each of which would
individually be subject to the vagaries of genetic drift [Olson
and Varki, 2003]. The above notwithstanding, many of the
(gene loss in humans)

nonsense mutations)

published examples of gene loss in the human lineage may well

have been neutral with respect to fitness, with the null allele
having simply become fixed through genetic drift.
One good example of adaptive pseudogenization is provided by
the human-specific inactivation of the caspase-12 (CASP12) gene.
The CASP12 gene has been specifically inactivated in humans by
a protein truncating transition but remains functional in other
mammals [Saleh et al., 2004]. Caspase 12 is thought to play an
important role in apoptosis and the processing of inflammatory
cytokines, including the interleukin-1 (IL-1) and nuclear factor
EGF-like module containing, mucin-like,

acetylneuraminic acid hydroxylase

(NF)-kB pathways [Nakagawa et al., 2000; Lamkanfi et al., 2002;

A disintegrin and metalloproteinase
domain 1 (fertilin a) pseudogene

Saleh et al., 2004]. The human-specific inactivating mutation has

Sialic acid binding Ig-like lectin 13

been identified in all Caucasians and Asians but has not yet been
Cytidine monophosphate-N-

fixed as a null-allele in humans since it is present in only
80% of

hormone receptor-like 4

Africans [Fischer et al., 2002; Saleh et al., 2004]. In about 20%

of people of African descent, the functional caspase 12 polypeptide
is still expressed, as it is in New World and Old World monkeys
and rodents [Saleh et al., 2004]. The full-length caspase-12
isoform encoded by the functional gene confers endotoxin
Haptoglobin

hyporesponsiveness, which appears to manifest in the clinic as

an increased susceptibility to severe life-threatening sepsis. By
contrast, possession of the truncated caspase-12 isoform is
associated with a reduced incidence of severe sepsis and
consequent mortality [Saleh et al., 2004]. By means of diversity
analyses of the region around the inactivating mutation, performed
to search for selective sweeps and to make coalescent simulations,
SIGLEC13
ADAM1

the drift towards the fixation of the CASP12 null allele has been
CMAH
EMR4

driven by positive selection [Wang et al., 2006; Xue et al., 2006].

HPP

The pseudogenization of the CASP12 gene appears to have

Human Mutation DOI 10.1002/humu

108 HUMAN MUTATION 28(2), 99^130, 2007
occurred prior to the out-of-Africa migration of modern humans
because the CASP12 T alleles of Africans and non-Africans share
the same origin [Wang et al., 2006]. Although CASP12 represents
the first reported case of adaptive gene loss in humans, the high
frequency of pseudogenization and examples of adaptive gene loss
in Drosophila [Takahashi et al., 2001; Greenberg et al., 2003, 2006]
suggest that this process may have generally played a key role
in evolution.
It is of course not only human-specific gene losses that will have
contributed to the genomic differences between humans and
chimpanzees; the inactivation or loss of genes in the chimpanzee
lineage is also likely to have played a role. Indeed, Mikkelsen
et al. [2005] identified a total of 53 human genes that were
either partially (17) or entirely (36) deleted in the chimpanzee.
According to the preliminary results of Wang et al. [2006], the
chimpanzee genome may contain even more pseudogenes than
that of humans, although the availability of a more refined
sequence of the chimpanzee genome will be required to draw firm
conclusions. It may be that some of the gene inactivation events
that originally occurred in incipient chimpanzee populations
were important in bringing about the permanent separation of
this lineage from that of early ancestral humans. Further in-
depth analyses of these gene losses in the chimpanzee are
needed to evaluate whether they occurred specifically in the
chimpanzee lineage or whether they actually represent
human-specific gains.
Interestingly, X-degenerated genes on the Y chromosome have
exhibited a significant level of gene decay during chimpanzee
evolution although not in the human lineage [Hughes et al.,
2005]. The causes and consequences of this lineage-specific
decay process are currently unknown but it has probably been
influenced by strong positive selection focused on other regions of
the chimpanzee Y chromosome [Hughes et al., 2005]. Since the Y
chromosome does not for the most part participate in recombina-
tion, selection must act on the chromosome as a unit. The genes
under positive selection on the chimpanzee Y chromosome could
be those specifically expressed in the testis and involved in
improving sperm competition [Rice, 1987]. It is not unreasonable
to assume that these genes could have evolved under powerful
selective pressure in view of the fairly promiscuous mating
behavior of extant chimpanzees [Gagneux et al., 1999].
EXPRESSION REGULATION AND
TRANSCRIPTOME EVOLUTION
Gene Expression Divergence Between Humans
and Chimpanzees
Changes in gene expression have had a major impact on the
evolution of eukaryotes [Wray et al., 2003] and hence probably
also on human gene evolution [King and Wilson, 1975; Carroll,
2005]. With the advent of DNA microarray technologies, it has
become possible to perform genomewide interhominoid trans-
criptome comparisons [Enard et al., 2002; Caceres et al., 2003;
Karaman et al., 2003; Marvanova et al., 2003; Uddin et al., 2004;
Khaitovich et al., 2005a; Gilad et al., 2006]. These analyses
have revealed many differences in the species- and tissue-specific
expression patterns of both humans and chimpanzees. Further,
microarray analyses have detected considerable diversity within
the human brain with respect to age-related changes in gene
expression. In the chimpanzee cortex, age-related changes in gene
expression have also been observed, but these patterns were
significantly different from those exhibited by the human cortex.
These findings imply the potential for the rapid evolution of
Human Mutation DOI 10.1002/humu
genomewide changes in gene expression associated with aging in
different species [Fraser et al., 2005].
The highest number of genes displaying differences in expres-
sion between human and chimpanzee was noted in the testis
[Khaitovich et al., 2005a]. By contrast, the lowest degree of gene
expression divergence between these species was observed in
the brain, probably due to the significant functional constraints
enforced upon this organ in higher primates [Enard et al.,
2002; Khaitovich et al., 2005a]. Remarkably, most of the genes
differentially expressed in the brain of humans as compared to
chimpanzees exhibit increased expression in humans [Enard et al.,
2002; Caceres et al., 2003; Gu and Gu, 2003; Hsieh et al., 2003].
The upregulation or induced expression in the human brain as
compared to chimpanzees would appear, however, to be brain-
specific, since in the liver no systematic increase or decrease in
the regulation of differentially-expressed genes in either humans or
chimpanzees was observed [Gilad et al., 2006]. In contrast to
earlier studies, these authors used species-specific microarrays for
five primate species including human and chimpanzee cDNAs to
avoid errors due to sequence divergence. They observed that most
genes are under selective pressure to maintain a constant level of
expression and that only a few genes (110/1,056) are differentially
expressed in humans as compared to chimpanzees. Among the
genes manifesting a higher expression level in humans, transcrip-
tion factors are themselves overrepresented, in contrast to
chimpanzees. Since transcription factors regulate gene expression,
these findings support the view that many differences between
humans and chimpanzees may be due to changes in gene
regulation [King and Wilson, 1975]. Remarkably, transcriptional
repressors of the KRAB-ZNF gene family are also characterized by
rapid expansion and lineage-specific diversification during primate
evolution [Hamilton et al., 2006].
It is always possible that measured mRNA levels may not reflect
the actual levels of functionally active proteins synthesized by
the respective genes [Gygi et al., 1999; Preuss et al., 2004].
Interestingly, parallel patterns of gene expression differences and
protein divergence have been observed in comparisons of human
and chimpanzee transcriptomes and proteomes [Bustamante
et al., 2005; Khaitovich et al., 2005a; Nielsen, 2006; Gilad
et al., 2006]. These parallel patterns indicate that significant gene
expression differences correlate with extensive divergence of the
encoded proteins. This suggests that both types of change have
evolved in concert and that similar factors may have influenced
protein and gene expression divergence [Khaitovich et al., 2005a].
Genes with tissue-specific expression patterns tend to display
the highest degree of divergence at both RNA and protein levels,
whereas genes that are expressed in several tissues tend to be the
least divergent between the species and to show the most minimal
expression differences [Khaitovich et al., 2005a]. This probably
reflects the greater selective constraints acting on genes that are
expressed in multiple tissues.
If these comparative analyses of gene expression and
protein divergence are complemented with data on intraspecies
variation (diversity) patterns, it should be possible to identify
genes that have evolved under positive selection. For example,
genes expressed in testis, and especially those located on
the X chromosome, display high expression and sequence
divergence but reduced diversity as compared to other tissues
such as liver, heart, and kidney [Khaitovich et al., 2005a]. This is
compatible with previous observations that genes encoding
proteins involved in reproduction tend to have evolved
under positive selection [Wyckoff et al., 2000; Swanson and
Vacquier, 2002].

Possible Causes of Gene Expression Divergence
The central question remains as to the molecular basis (and its
ultimate causes) of the observed gene expression divergence
between humans and chimpanzees. The processes involved are
likely to include the sequence divergence of regulatory
regions, differences in the control of transcriptional initiation,
RNA processing and translation, the modification of chromatin
structure, and potentially also differences in DNA methylation.
Sequence variation in gene promoter regions has certainly
been shown to influence transcription levels in individual
humans [Hoogendoorn et al., 2003; Bray et al., 2003; Trinklein
et al., 2003; Buckland, 2004], probably due to the consequent
differential binding of cis- and trans-acting regulatory factors
[Pilpel et al., 2001]. In the same vein, nucleotide sequence
divergence is also likely to be responsible for differentially modulat-
ing promoter activity in humans as compared to chimpanzees,
one example being the apolipoprotein(a) (LPA) gene promoter
[Huby et al., 2001].
A general tendency has been noted for the 50 upstream
noncoding regions of genes to exhibit higher rates of nucleotide
sequence divergence between humans and chimpanzees than in
coding sequences and 30 UTRs [Chen and Li, 2001; Hellmann
et al., 2003a; Shi et al., 2003a; Keightley et al., 2005]. However,
the determination of the overall divergence of promoter regions is
probably always going to be of limited utility since comparatively
few nucleotide changes are likely to exert a strong influence on
gene expression, irrespective of the overall level of sequence
divergence. In support of this view, the comparative analysis of
16-bp blocks from promoter regions of 5,547 human-chimpanzee
orthologous gene pairs has indicated that small sequence windows
have evolved under selective constraints and that purifying
selection has indeed been operating within hominid noncoding
sequences [Bush and Lahn, 2005]. Further, human/chimpanzee/
mouse comparisons of candidate transcription factor binding sites
has revealed that the loss of such sites in the human lineage has
not simply been random and that these changes in regulatory
motifs have affected the expression of specific functional categories
of genes, involving principally the olfactory system [Donaldson
and Gottgens, 2006].
The unambiguous identification of relevant regulatory elements
could facilitate the design of functional assays that might then
be used to identify the molecular basis of expression divergence
between humans and chimpanzees. In practice, however, the
design of such assays has proved to be very difficult. Indeed,
comparative in vitro expression studies using promoter sequences
from genes that are differentially expressed in these species suggest
that there is no simple relationship between in vitro promoter
activity in cell lines and expression divergence in tissues in vivo
[Heissig et al., 2005]. It necessarily follows that expression
divergence will be difficult to predict by in vitro expression/
DNA sequence analysis, probably as a consequence of the tissue-
and cell linespecificity of the transcription factors involved and
their potentially limited availability in vitro [Heissig et al., 2005].
Just as gene expression diversity between individual humans is
known to be influenced by multiple cis- and trans-acting factors
[Morley et al., 2004; Stranger et al., 2005], so it is likely that
multiple genetic differences will be found to influence expression
divergence between humans and chimpanzees. Particularly inter-
esting in the context of the expression divergence between these
higher primates is the finding that transcription factors have
evolved rather rapidly in the human lineage by comparison
with other human proteins [Bustamante et al., 2005]. The
HUMAN MUTATION 28(2), 99^130, 2007 109
accelerated evolution of transcription factors is indicative of how
a relatively small number of genetic changes in key locations may
pleiotropically affect the expression patterns of a myriad of
different genes by influencing their transcription factor binding
capacity. We may therefore surmise that the accelerated evolution
of transcription factors in the human lineage has in all likelihood
had a major impact on gene expression divergence between
human and chimpanzee.
When humans and chimpanzees have been compared with
respect to DNA methylation patterns, the degree of CpG
methylation has generally been found to be higher in the human
brain as compared to chimpanzee brain. Higher levels of CpG
methylation are also apparent in other human tissues compara-
tively investigated, such as liver and lymphocytes, but are not
as pronounced as those noted for the brain. There is, however,
no evidence as yet that these changes in methylation status
are associated with the observed expression divergence [Enard
et al., 2004].
Adaptive Evolution of Transcriptional Regulation
Although our knowledge of the evolution of transcriptional
cis-regulation is still rather limited and will probably be very
difficult to investigate on a large scale [Huby et al., 2001; Heissig
et al., 2005], the population genetic and phylogenetic evidence
for positive selection acting on regulatory mutations at the human
F7 [Hahn et al., 2004] and PDYN [Rockman et al., 2005] loci
imply that positive selection can operate on regulatory mutations
that have occurred specifically in the human lineage. One example
of this phenomenon is provided by the PDYN gene, which encodes
prodynorphin, a precursor molecule for a series of endogenous
opioids and neuropeptides with critical roles in the anticipation
and experience of pain perception, behavior including social
attachment and bonding, as well as learning and memory [Rodgers
and Cooper, 1988; Wagner et al., 1993; Cheng et al., 2002; Moles
et al., 2004]. A 68-bp tandem repeat polymorphism in the human
PDYN gene promoter, 1,250 bp upstream from the transcriptional
start site, influences the inducibility of the gene [Zimprich et al.,
2000]. During human evolution, a duplication of this 68-bp
regulatory element occurred generating four different genotypes
containing either one, two, three, or four copies of the repeat. By
contrast, great apes and Old World monkeys have only one copy
of the 68-bp element. Interestingly, this element has evolved
rapidly in the human lineage, as indicated by the five nucleotide
substitutions that occurred after the separation of humans and
chimpanzees, but prior to the human-specific duplication of the
element. This number of nucleotide substitutions is somewhat
higher than that expected for a neutrally evolving segment and
suggests that the rapid evolution of the human 68-bp repeat
occurred under positive selection [Rockman et al., 2005]. The
combined effect of these fixed cis-regulatory mutations would
appear to be the upregulation of PDYN gene expression upon
induction by intracellular calcium release. An excess of high-
frequency derived mutations in the flanking regions of the 68-bp
segments suggests that the entire region, including the regulatory
element, evolved under positive selection. Further, the pattern of
microsatellite variation within and between modern human
populations implies that recent selection, subsequent to the
fixation of the human-specific mutations, has favored different
PDYN cis-regulatory alleles in different parts of the world. Thus,
selection, both ancient and recent, has served to highlight the
significance of PDYN gene expression to human biology. Although
the direct target of this selection is unclear, it appears to involve
Human Mutation DOI 10.1002/humu
110 HUMAN MUTATION 28(2), 99^130, 2007
changes in the inducibility of prodynorphin, an endogenous opioid
precursor that is a prime candidate for involvement in conferring
human-specific traits due to its role in perception, emotion,
nociception, and learning [Rodgers and Cooper, 1988; Wagner
et al., 1993; Cheng et al., 2002; Moles et al., 2004; Balter, 2005].
In accordance with the predictions of the regulatory hypothesis
put forward by King and Wilson [1975], the evolutionary history
of the PDYN gene suggests that it may represent a good example of
a gene in which regulatory changes have played a significant role
in human evolution. It remains to be seen how frequently
regulatory changes fixed in the human lineage have been adaptive,
having evolved under selective pressure as opposed to being fixed
simply as a consequence of stochastic processes.
Khaitovich et al. [2004, 2005b] have presented a model, which
proposes that the majority of expression differences observed
between species are selectively neutral and lack any functional
significance. Consistent with this postulate, only a very small
proportion of the gene expression changes in human relative to
chimpanzee appear to be due to diversifying selection [Hsieh et al.,
2003]. However, such a neutralist model would appear to be at
odds with: 1) the upregulation of gene expression that is specific
to the human brain and not apparent either in the chimpanzee
brain or in the liver of humans and chimpanzees [Gu and
Gu, 2003; Gilad et al., 2006]; and 2) the rapid evolution of
primate transcription factors [Gilad et al., 2006]. Among possible
adaptive scenarios, it may be that the general level of physiological
activity in the brain, and in particular the cerebral cortex, is higher
in humans than in chimpanzee with the higher levels of gene
expression in humans being required to meet the correspondingly
higher requirements for brain proteins. Clearly, further studies are
needed to explore to what extent the higher gene expression levels
characteristic of the human brain are reflected in the levels of their
protein products [Preuss et al., 2004].
ALTERNATIVE SPLICING
Alternative splicing contributes very significantly to increased
transcriptome and proteome diversity as well as to interspecies
divergence [Boue et al., 2003].
An estimated 60% of all human genes employ alternative
splicing [Mironov et al., 1999; Lander et al., 2001]. Dual coding
regions in particular are characterized by alternative splicing; the
same exon sequences are shared by different transcripts encoding
distinct amino acid sequences in different reading frames. At least
7% of all alternatively spliced genes in the human genome contain
multiple coding regions [Liang and Landweber, 2006]. Compara-
tive analysis of orthologous sequences in the chimpanzee, mouse,
rat, dog, and chicken genomes has indicated that most secondary
reading frames in dual coding regions have originated during
mammalian evolution. Interestingly, 4% of human dual coding
regions are not conserved in chimpanzees, as indicated by the
presence of stop codons that disrupt one of the two reading frames
[Liang and Landweber, 2006].
Dual coding genes can be grouped into two categories: if the
dual reading frame is intact, it may be regarded as the ancestral
reading frame (ARF), whereas the presence of a stop codon in
one of the two reading frames defines it as the derived
reading frame (DRF). Comparative analysis between human
and chimpanzee has revealed that the reading frames associated
with the original splicing patterns (ARFs) have acquired
considerably fewer substitutions than the derived ones, suggesting
that ARFs have been maintained by stronger selection pressure
[Liang and Landweber, 2006]. This observation is in accord with
Human Mutation DOI 10.1002/humu
the finding that alternatively spliced transcripts, conserved
between human and mouse, have evolved under strong selection
[Modrek and Lee, 2003]. Further, it suggests that alternative
splicing may have played a major role in genome evolution because
new exons will have been able to evolve with fewer constraints.
Alternative splicing patterns observed in multiple species may
be considered to be ancestral and such cases usually display
evidence of strong functional constraints. Frequently, these
exons are modular in that their lengths are multiples of three
nucleotides [Thanaraj et al., 2003; Resch et al., 2004] thereby
allowing their alternative inclusion or exclusion without disrupting
the reading frame which would lead and leading to premature
protein truncation.
When human and chimpanzee exon flanking regions were
compared, a significant reduction in the nucleotide substitution
rate of the flanking regions of alternatively spliced exons was
observed, independent of the site-by-site variation in mutability
due to different CpG contexts. Thus, increased purifying selection
appears to have impacted on exon flanking regions and thus, in all
likelihood, the regulation of alternative splicing in humans and
chimpanzees [Xing et al., 2006].
A rather specific type of alternative splicing, the differential
exclusion of exons due to pseudoexonization, may also have
contributed to the genetic divergence between humans and
chimpanzees. An example of this phenomenon is provided by
the glycophorin B (GYPB) gene, in which exon 3 is expressed
in chimpanzees but silenced as a pseudoexon in humans [Huang
et al., 1995].
CHROMOSOMAL DIFFERENCES AND VARIATION
ACROSS CHROMOSOMES
Molecular cytogenetic analyses of hominoids have indicated that
humans and orangutans have retained karyotypes closer
to the putative ancestral hominoid karyotype than have the African
great apes (chimpanzee and gorilla), which display more lineage-
specific derived structural rearrangements [Dutrillaux, 1979; Muller
and Wienberg, 2001]. The chimpanzee and human genomes may
be distinguished by 10 karyotypic differences that include nine
pericentric inversions and one fusion that gave rise to human
chromosome 2 [Yunis and Prakash, 1982; Gross et al., 2006]. Most
of these rearrangements have now been fully characterized by
analysis of the breakpoint regions; in none of these cases, however,
has a gene been interrupted by the breakpoints [IJdo et al., 1991;
Nickerson and Nelson, 1998; Marzella et al., 2000; Fan et al., 2002;
Kehrer-Sawatzki et al., 2002, 2005a,b,c; Locke et al., 2003a;
Dennehey et al., 2004; Goidts et al., 2004, 2005; Shimada et al.,
2005; Szamalek et al., 2005, 2006a]. Segmental duplications were
identified in the breakpoint regions of six of the nine pericentric
inversions, suggesting that these duplicates could have mediated or
at least facilitated the inversions. Interestingly, human-specific
segmental duplications were identified at the breakpoints of both
the chromosome 1 and 18 inversions, hinting that these sequence
acquisitions could have rendered the respective breakpoint regions
susceptible to homologous recombination [Goidts et al., 2005;
Szamalek et al., 2006a].
Seven of the large pericentric inversions appear to have been
fixed in the chimpanzee lineage (Supplementary Table S3) and
comparative breakpoint analyses have shown that the two
sister species, the common chimpanzee (Pan troglodytes) and the
pygmy chimpanzee (Pan paniscus) share the same pericentric
inversions [Locke et al., 2003a; Szamalek et al., 2006b]. Thus,
these inversions must have predated the separation of the two
 HUMAN MUTATION 28(2), 99^130, 2007 111

chimpanzee species
0.862 Mya [Yoder and Yang, 2000; Won Alu Sequence Insertions/Deletions
and Hey, 2005]. It is as yet unclear whether any of these
With up to 106 copies in the human genome, Alu sequences
rearrangements played a role during the process of speciation of
constitute the most frequently encountered type of short
humans and chimpanzees. According to the recombination
interspersed repeat element (SINEs). Alu mobilization has, how-
suppression model, these chromosomal rearrangements, in their
ever, proceeded at different rates in the human and chimpanzee
heterozygous state, could have acted as a partial barrier to gene
lineages. Thus, comparison of chimpanzee chromosome 22 and its
flow between early populations of emerging humans and
human homolog, chromosome 21, revealed a two-fold greater
chimpanzees at a time when interbreeding was still possible
frequency of Alu insertions in humans as compared to the
[Navarro and Barton, 2003; Rieseberg and Livingstone, 2003].
chimpanzee. Moreover, the comparison of human and chimpanzee
To test this hypothesis, Navarro and Barton [2003] analyzed 115
Y chromosomes has revealed higher Alu element retrotransposi-
genes and observed that the DNA sequence divergence on
tional activity in the human lineage [Hughes et al., 2005].
rearranged chromosomes was slightly higher than on colinear
However, by analogy with the higher rate of nucleotide sequence
chromosomes. Furthermore, the rate of protein evolution on
diversity in chimpanzees, the Alu sequence diversity (i.e., presence
rearranged chromosomes as measured by KA/KS ratios was twice
or absence of Alu elements at specific genomic locations within a
that observed on colinear chromosomes [Navarro and Barton,
given species) is 1.7 times higher in the chimpanzee as compared
2003]. These findings were therefore consistent with the view that
to human [Hedges et al., 2004].
large chromosomal rearrangements could have led to reduced gene
An expansion in the overall size of the human genome due to a
flow and accelerated adaptive evolution. However, reexamination
higher rate of Alu retrotranspositional activity in the human
of this hypothesis using larger datasets did not confirm these
lineage has been detected by Liu et al. [2003]. They compared
findings and failed to provide support for the hypothesis that gross

10.6 Mb of genomic sequence from lemur, baboon, chimpanzee,
chromosomal rearrangements affected the rate of genetic diver-
and human and noted a marked paucity of chimpanzee Alu inser-
gence between humans and chimpanzees [Lu et al., 2003; Zhang
tions as compared to human. Since the separation of chimpanzees
et al., 2004a; Vallender and Lahn, 2004b].
and humans from their common ancestor 5 to 7 Mya,
7000 Alu
During the course of their comprehensive comparative analysis,
elements have been fixed in the human genome including the
the CSAC also investigated the variation in protein divergence
human-specific subfamilies AluYa5 and AluYb8 [Carroll et al.,
across different chromosomes [Mikkelsen et al., 2005]. This re-
2001; Carter et al., 2004; Gibbons et al., 2004; Otieno et al., 2004;
examination did not, however, reveal any evidence for the
Mikkelsen et al., 2005; Mills et al., 2006]. Genomewide
accelerated evolution of gene products encoded by genes on
comparisons have indicated that the number of Alu insertions in
chromosomes with major rearrangements. Nevertheless, the large
humans has been
3.4-fold higher than in the chimpanzee and
inversions appear to be associated with an increase in interspecies
that the distributions of these elements among various Alu
differences in brain gene expression when genes on rearranged vs.
subfamilies also differed between the two species [Mills et al.,
colinear chromosomes are compared. Thus, the rearrangements
2006] (Supplementary Fig. S2). AluYa5, AluYb8, AluY, and
may have had indirect effects on the expression of linked genes
AluYc1 are highly abundant in humans, whereas only AluYc1
[Marques-Bonet et al., 2004].
and AluY are abundant in chimpanzees [Mills et al., 2006]. Clearly,
significantly more Alu retrotransposition has occurred in the
human lineage and a different set of Alu elements has been
DYNAMICS OF STRUCTURAL amplified in humans as compared to chimpanzees (Fig. 2).
DIFFERENCES BETWEEN THE HUMAN
AND CHIMPANZEE GENOMES
Insertions/Deletions 10000000

1000000
Recent progress in comparing the human and chimpanzee
Number of rearrangements

genomes has revealed that the structural divergence between 100000

them is not confined to gross chromosomal rearrangements but 10000

also includes a considerable number of submicroscopic structural 1000
differences [Britten, 2002; Britten et al., 2003; Frazer et al.,
100
2003; Liu et al., 2003; Fortna et al., 2004; Newman et al.,
10
2005; Mikkelsen et al., 2005; Kuroki et al., 2006]. These
include insertions and deletions that have considerably altered 1

the landscape of the human and chimpanzee genomes

after the separation of both lineages. Indeed, insertions of modest
f

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size (1 bp to 15 kb) have given rise to a total of
32 Mb of

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human-specific DNA sequence by comparison with the chimpan-

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zee genome [Mikkelsen et al., 2005] and a comparable figure for

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chimpanzee-specific sequences may reasonably be assumed.

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Nu

The genomewide comparison performed by the CSAC has

provided comprehensive information about the nature and
frequency of lineage-specific insertions and deletions. Most inser-
tions/deletions are small, with 96% being o20 bp and 98.6% being
o80 bp (Fig. 1). Approximately 1.4% of all modestly-sized
insertions/deletions are larger than 80 bp; these involve a variety
of different sequences but the majority comprise satellites, micro-
satellites, high copy number repeats, and transposons.
Length of the rearrangement
FIGURE 1. Graphical representation of the genetic and cytoge-
netic dierences between the human and chimpanzee genomes.
Rearrangements are ordered by size (x-axis). The number of
rearrangements is represented on a logarithmic scale (y-axis).
a
Mikkelsen et al. [2005]; bChen and Li [2001]; cBritten [2002];
d
Feuk et al. [2005]; eCheng et al. [2005]; f Yunis and Prakash
[1982].

Human Mutation DOI 10.1002/humu

112 HUMAN MUTATION 28(2), 99^130, 2007
FIGURE 2. Number of transposon insertions within genes, in
human and chimpanzee genomes, according to Mills et al. [2006].
The majority of these insertions areAlu, L1, and SVA elements.
In contrast to the expansion of repetitive sequences such as
retroelements, the deletion of these sequences has also been
observed in a lineage-specific manner. An estimated 0.5 to 1%
of the apparent retroelement insertions that distinguish humans the human endogenous retrovirus K (HERV-K) HERV-K10 [Ono
from chimpanzees actually represent deletions in one or other
genome; this particularly affects members of the older Alu sub-
families, such as AluS and AluJ, which are common to both
humans and chimpanzees. These deletions have probably been
mediated by recombination between 10 to 20 bp direct repeats
flanking the retroelements [van de Lagemaat et al., 2005].
Recombination between Alu elements is considered to be the
mechanism underlying the 492 human-specific deletions between
101 and 7,255 bp. These deletions also affected exons and resulted
in the loss of 400 kb from the human genome as compared to the
chimpanzee [Sen et al., 2006]. Thus, Alu-mediated deletions have
counteracted, at least in part, the increase in the size of the human
genome due to Alu insertions. Both processes have clearly
contributed to the evolution of the human genome.
LINE Element Insertions
Long interspersed elements (LINE-1 or L1) comprise
17%
of the human genome sequence and are the most abundant
and the only active autonomous non-long terminal repeat (LTR)
retrotransposons in the human genome. However, of 4500,000
L1 copies, only
80 to 100 are capable of active retrotransposition
[Brouha et al., 2003].
Some L1 retrotransposons have been specifically amplified since
the origin of the primates [Salem et al., 2003; Vincent et al., 2003;
Ho et al., 2005]. Two phases of L1 expansion may be discerned,
during which different L1 families experienced differing degrees of
replicative success. The most intense period of L1 activity involved
families L1PA8L1PA3 and lasted from
40 to 12 Mya [Khan
et al., 2006]. Their expansion was associated with the amplifica-
tion of AluY elements and many processed pseudogenes [Batzer
and Deininger, 2002; Ohshima et al., 2003]. Approximately 2,000
L1 elements have become inserted specifically into the human
genome since human-chimpanzee divergence, a process which
may have added as much as 1.3 Mb of DNA to the human genome
over the last
5 Myrs [Han et al., 2005; Mikkelsen et al., 2005].
This expansion may in part account for the larger genome sizes
of anthropoid primates (e.g., monkeys, apes, and human) as compared
with the prosimians (e.g., lemurs and galagos) [Liu et al., 2003].
These L1 expansions, however, occurred prior to the separation of
hominoids. Unlike the human lineage-specific Alu expansion, the
rate of L1 element retrotranspositional activity has been relatively
similar in the genomes of human and chimpanzee; they both possess
Human Mutation DOI 10.1002/humu

2,000 lineage-specific L1 elements [Mikkelsen et al., 2005]. This
contrasts with earlier assertions of a higher rate of L1 insertion in
the chimpanzee lineage [Mathews et al., 2003; Mills et al., 2006].
However, chromosome-specific differences may be assumed, since
on the chimpanzee Y chromosome, insertions of L1 elements and
endogenous retroviruses have been significantly more frequent
than on the human Y chromosome [Hughes et al., 2005].
Finally, L1 insertions into the human genome are often
accompanied by target site deletions. Han et al. [2005] have
characterized 50 such L1 insertion-mediated deletions in the
human and chimpanzee genomes resulting in the loss of a total of

18 kb from the human genome and
15 kb from the chimpanzee
genome.
SVA Elements
The youngest family of primate retrotransposons that arose after
the separation of Old World monkeys from the hominoid ancestral
lineage are the SVA elements, termed according to their main
components: a SINE-R sequence derived from an LTR element of
et al., 1987], VNTR, a central variable number tandem repeat
that is rich in CpG sequences, and a partial Alu-sequence in
reverse orientation [Shen et al., 1994b]. SVA elements end with a
poly (A) tail and are flanked by target site duplications similar to
those that flank Alu and L1 elements [Ostertag et al., 2003;
Bennett et al., 2004]. SVA elements are frequently polymorphic
among humans [Bennett et al., 2004] and some instances have
been reported of SVA insertions causing disease [Kobayashi et al.,
1998; Ostertag et al., 2003].
Six SVA subfamilies have been identified and exhibit consider-
able lineage-specific activity in humans and chimpanzees, sprout-
ing two human-specific subfamilies [Wang et al., 2005]. Direct
genome comparisons have revealed
1,000 SVA copies specific
to the human lineage and a comparable number of chimpanzee
lineage-specific integrations [Mikkelsen et al., 2005]. Full-length
SVA elements have a high G1C content (about 60%) and
contain transcription factor SP1 binding sites. This confers upon
SVAs the potential to mobilize CpG islands and hence their
insertion could influence the expression of flanking genes.
Mikkelsen et al. [2005] identified human-specific SVA inser-
tions near the promoters of three genes: MRGX3 (G protein-
coupled receptor MRGX3), SLC2A11 (glucose transporter protein
10), and SULT1B1 (sulfotransferase family cytosolic 1B). Detailed
analyses are, however, necessary to confirm or exclude effects of
the SVA integration with respect to the expression of the
respective genes.
Remarkably, a large number of lineage-specific transposon
insertions have occurred within genes (2,642 in humans and
990 in chimpanzees; Fig. 2). In view of the extent of these
insertional events, it is very likely that at least some of them have
influenced gene expression [Mills et al., 2006]. Since the number
of transposon insertions has been higher in the human lineage, we
surmise that the impact of transposon-mediated expression
differences is likely to have been more pronounced in humans.
Endogenous Retroviral Elements
Endogenous retroviruses and LTRs comprise
8% of the human
genome. They arose early during primate evolution by infection
and integration of exogenous source viruses and were subsequently
amplified by retrotransposition of preexisting endogenous
retroviruses. The HERV-K subfamily is the most conserved
evolutionarily and is capable of forming virus-like particles [Costas,

2001; reviewed in Bannert and Kurth, 2004]. About 30 to 50
HERV-K proviruses are found in the human genome; the youngest
known HERV-K provirus, HERV-K113, has an estimated age of
o200,000 years [Turner et al., 2001]. Divergent patterns of recent
retroviral integrations into the human and chimpanzee genomes
have been observed [Buzdin et al., 2003; Jern et al., 2006;
Polavarapu et al., 2006]. Comparing the human and chimpanzee
genomes, 45 chimpanzee-specific insertions (one full-length
and 44 solo LTRs) and 73 human-specific insertions (seven full-
length and 66 LTRs) have been identified corresponding to
lineage-specific retrotranspositional events [Mikkelsen et al.,
2005; Romano et al., 2006]. Finally, a genomewide survey of
endogenous retroviral positional variation between humans and
chimpanzees has shown that 7% of all human-chimpanzee
insertion/deletion variation is associated with endogenous retro-
viral sequences [Polavarapu et al., 2006].
These insertions could have influenced the expression of nearby
genes by virtue of the presence of their internal polyadenylation
signals and promoter and enhancer sequences, as well as cryptic
splice sites. Indeed, Yohn et al. [2005] have characterized the
insertion sites of a PTERV1 retroviral element, sequences which
are specific to the African great apes including the chimpanzee but
that are not found in human. Of 10 chimpanzee genes found to
harbor intronic PTERV1 elements, six exhibit significant differ-
ences in gene expression by comparison with human (in five of
these, gene expression is lower in the chimpanzee).
Microsatellite and Triplet Repeat Expansion
The tendency of the human genome to expand in size is also
reflected in microsatellite dynamics. Microsatellites are iterations
of short (16 bp) sequence motifs, with the length of the repeats
being generally less than 30 bp. Comparative analyses of micro-
satellite length in humans and chimpanzees has revealed that
human microsatellites are longer than their orthologous chimpan-
zee equivalents, even when the effects of ascertainment bias due to
microsatellite length are allowed for [Cooper et al., 1998; Vowles
and Amos, 2006]. This suggests that expansion mutations have
occurred disproportionately in the human lineage. Microsatellite
evolution has, however, turned out to be quite heterogeneous.
Human dinucleotide repeats tend to be longer than their orthologs
in chimpanzees, whereas the opposite trend is apparent in
mononucleotide repeat arrays. Furthermore, the rate of divergence
between orthologous microsatellites is significantly higher at longer
loci, which also display evidence of higher mutability per repeat
number. Thus, locus-specific mutation rate variability between
species suggests that microsatellite evolution is a highly dynamic
process [Webster et al., 2002].
Remarkably, triplet repeats within coding regions also seem to
be longer in humans than in chimpanzees as observed for GAA
triplet repeat-containing loci [Clark et al., 2006], cancer-related
genes [Puente et al., 2006], and genes associated with neurode-
generative disorders [Choong et al., 1998; Andres et al., 2004b].
With respect to the latter class of genes, not only do humans
possess larger alleles as compared with the great apes, but they also
display greater length variability, a finding that is suggestive of a
relationship between the level of variability and expansion
potential [Andres et al., 2004b].
Poly-purine/pyrimidine sequences (RY tracts) are able to form
triplex DNA structures that can block DNA replication and
interfere with transcription. Such non-B DNA conformations are
highly mutagenic due to their propensity to induce double strand
breaks, and are frequently found at the breakpoints of gross
HUMAN MUTATION 28(2), 99^130, 2007 113
deletions and other rearrangements (summarized in Bacolla and
Wells [2004]; Bacolla et al. [2004, 2006]). Detailed comparison of
long RY tracts in human and mouse as well as in the human and
chimpanzee genomes have indicated that they have mutated
much faster than their flanking sequences due to mutational
mechanisms such as replication slippage and recombination as well
as nucleotide substitution. Furthermore, 80% of the pure RY tracts
4250 bp are longer in humans than in chimpanzees, while the
length divergence of RY tracts in the human as compared to the
chimpanzee genome is significantly higher than the average rate of
nucleotide substitution in both genomes [Bacolla et al., 2006].
Gross Insertions and Duplications
Human/chimpanzee genome comparisons have also revealed a
considerable number of human-specific insertions of larger size
(415 kb). These frequently contain genes (or partial gene
sequences) and have given rise to
8 Mb of human-specific
sequences and probably a similar amount of chimpanzee-specific
sequence [Mikkelsen et al., 2005]. Taken together, some 40 to
45 Mb of lineage-specific sequence results from insertions/
deletions. Thus, a total of
90 Mb, the sum of all lineage-specific
sequences, contribute to the divergence evident between the
human and chimpanzee genomes [Mikkelsen et al., 2005].
The total sequence divergence with respect to the number of
nucleotides affected by deletions and insertions is thus some three-
fold higher than the divergence due to nucleotide substitutions.
However, the number of underlying mutational events is clearly
much lower for insertions/deletions than for nucleotide substitu-
tions (Fig. 1).
It may be that the number of lineage-specific insertions/deletions
detected by the CSAC using direct genome alignments was
underestimated owing to the draft version status of the chimpanzee
genome. An alternative approach to investigating the extent of
structural divergence between the human and chimpanzee genomes
has involved the mapping of fosmid end-pairs of a chimpanzee
library against the human reference sequence. By these means, a
total of 477 insertions/deletions 412 kb were identified [Newman
et al., 2005]. These structural variants encompass a total of
424 Mb DNA and include more than 200 genes whose structure
and/or expression could consequently be perturbed.
In addition to direct sequence comparisons, new methodologies
such as array comparative genomic hybridization (aCGH) using
genomic clones have proved to be capable of detecting copy-
number differences (CNDs) of 40 kb up to several Mb between the
human and chimpanzee genomes [Locke et al., 2003b; Goidts
et al., 2006a; Wilson et al., 2006a]. These CNDs represent
deletions and duplications, also termed intermediate sized
rearrangements since they are not visible by G-banding analysis
of chromosomes in contrast to the cytogenetically visible gross
rearrangements such as the nine pericentric inversions and the
fusion that gave rise to human chromosome 2. The majority of
these CNDs map to regions of segmental duplication, indicating
that this fraction of the genome is a major source of the variability
evident between humans and chimpanzees.
A genomewide comparison of segmental duplications between
the two primate species has been performed by Cheng et al.
[2005]; although
66% of the autosomal sequence duplicated
in humans was also found to be duplicated in the chimpanzee,
a considerable fraction (33%; encompassing 26.5 Mb) was
duplicated only in the human lineage [Cheng et al., 2005]. These
human-specific duplication intervals map to at least 500 distinct
regions (of average length
55 kb) distributed throughout the
Human Mutation DOI 10.1002/humu
114 HUMAN MUTATION 28(2), 99^130, 2007
genome. By contrast, only
17% (11.4 Mb) of the duplicated
sequences in the chimpanzee are chimpanzee-specific in that they
are not duplicated in the human genome [Cheng et al., 2005].
Segmental duplications have thus shaped the architecture of the
human genome [Eichler et al., 1996; Trask et al., 1998; Bailey
et al., 2002a,b; Armengol et al., 2003; Cheung et al., 2003;
Bailey et al., 2004; She et al., 2004; Stankiewicz et al., 2004;
Zhang et al., 2005b; Bailey and Eichler, 2006] and comparison
with the chimpanzee genome indicates that this has occurred in a
lineage-specific manner [Cheng et al., 2005; Goidts et al., 2006a;
Wilson et al., 2006a; Wooding and Jorde, 2006]. In view of the
enrichment of genes within segmental duplications, this is likely to
have had quite important implications for the emergence of new
genes and gene expression differences between the species.
SEGMENTAL DUPLICATIONS AND STRUCTURAL
DIFFERENCES IN SUBTELOMERIC REGIONS
BETWEEN HUMANS AND CHIMPANZEES
Segmental duplications (also termed low copy repeats [LCRs])
are implicated in the genesis of disease-associated rearrangements
owing to their inherent propensity to nonallelic homologous
recombination, the molecular basis of a wide range of so called
genomic disorders. The latter are caused by rearrangements
in genomic regions with complex architecture [reviewed in
Lupski, 1998; Ji et al., 2000; Shaw and Lupski, 2004; Lupski
and Stankiewicz, 2005]. Analysis of the human genome assembly
version 15 revealed that
4% of the human genome is covered by
sequence duplications and the extent of segmental duplication
varies from 1 to 14% between the 24 chromosomes.
Intrachromosomal duplications are more frequent than inter-
chromosomal duplications (Supplementary Fig. S3; Zhang et al.
[2005b]) and a peak of intrachromosomal duplication activity
appears to have occurred in the ancestral hominoid genome

10 Mya. By contrast, interchromosomal duplication dispersal
peaked somewhat earlier, during or after the separation of the
Old World monkeys from the hominoid lineage
25 Mya [She
et al., 2006]. Segmental duplications are enriched in pericen-
tromeric and subtelomeric regions of human chromosomes [Bailey
et al., 2002a; Zhang et al., 2005b]. The dynamics of duplicative
transposition are significantly more prominent in these regions by
comparison with the genome average [Mefford and Trask, 2002;
She et al., 2004; Linardopoulou et al., 2005]. One consequence of
this is the extensive polymorphism present in human subtelomeric
regions [Wilkie et al., 1991; Trask et al., 1998; Daniels et al.,
2001], which possess a high concentration of genes [Flint et al.,
1997; Saccone et al., 1992].
A considerable number of structural differences are evident
between the subtelomeric regions of human and chimpanzee
chromosomes using classical cytogenetic techniques. In contrast to
humans, chimpanzees and gorillas possess remarkable amounts of
additional positive G-banded chromatin at the chromosomal ends
[Yunis and Prakash, 1982]. These telomeric caps contain arrays
of a 32-bp AT-rich repeat sequence that is abundant within the
telomeric regions of the African great apes but absent from the
human genome [Royle et al., 1994]. In these subtelomeric regions
of chimpanzee chromosomes, massive expansions of one specific
segmental duplication occurred, leading to the generation of an
additional 16 Mb of genomic DNA that is not present in humans
[Cheng et al., 2005]. In addition to the subterminal portions of
chimpanzee chromosomes, the hyperexpansion of these sequences
also occurred in interstitial regions of chromosomes VII and XIII.
Sequence analysis of one chimpanzee locus corresponding to these
Human Mutation DOI 10.1002/humu
hyperexpanded sequences has indicated the presence of a single
copy of a 36-kb segmental duplication and a 14.5-kb cluster of the
aforementioned 32-bp repeats [Cheng et al., 2005]. Subtelomeric
regions of chimpanzee chromosomes would thus appear to have
been susceptible to duplicative transposition, expansion of repeats,
and segmental duplications, leading to an asymmetrical increase in
duplicated DNA in the chimpanzee lineage as compared to
humans [Cheng et al., 2005].
Comparative analyses have revealed further significant differ-
ences between the human and chimpanzee genomes caused by
copy number differences in subtelomeric segments [Trask et al.,
1998; Monfouilloux et al., 1998; Martin et al., 2002]. Human
subtelomeric regions were massively rearranged after divergence
from the chimpanzee lineage with at least 49% (1.13 Mb) of
human subtelomeric sequences being generated after the split
of both lineages [Linardopoulou et al., 2005]. Repeated trans-
locations between chromosomal ends then gave rise to complex
segmental duplications and at least one-half of the known subtelo-
meric sequence in humans has been generated by interchromo-
somal sequence transfer between segmental duplications after the
divergence of humans and chimpanzees [Linardopoulou et al.,
2005]. Subtelomeric, pericentromeric, and centric regions of
chromosomes constituted preferred acceptor sites of duplica-
tive transposition during the evolution of the human and
chimpanzee genomes, although to different degrees and with
differing preferences of genomic locations [Fortna et al., 2004;
Cheng et al., 2005; Mikkelsen et al., 2005].
EVOLUTION OF HUMAN CENTROMERES
The centromeres of human chromosomes are extremely
specialized loci that are critically important for orderly chromo-
some segregation. Their conserved function contrasts, however,
with the rapid evolution of their component DNA sequences
[reviewed by Henikoff et al., 2001; Henikoff and Dalal, 2005].
Normal human centromeres contain huge amounts of a-satellite
sequence associated with centromere function and kinetochore
assembly [Schueler et al., 2001; Ikeno et al., 1998; Grimes et al.,
2002; Spence et al., 2002; Porter and Farr, 2004] and specific
to the primate lineage [Willard, 1990; Alves et al., 1994]. These
sequences exist in two different forms, the higher-order and the
monomeric a-satellites, which evolved by complex patterns of
inter- and intrachromosomal exchanges [Warburton and Willard,
1996; Alexandrov et al., 2001; Rudd and Willard, 2004]. Higher-
order a-satellite DNA comprises homogeneous 3- to 5-Mb
arrays of repeat units in tandem orientation [Wevrick and Willard,
1989; Lee et al., 1997; Mahtani and Willard, 1998]. Monomeric
a-satellites, however, lack detectable higher-order periodicity, and
the monomers are more heterogeneous than the higher-order
repeat units [Rudd and Willard, 2004]. In normal human
centromeres, monomeric a-satellites flank the large array of
higher-order a-satellites [Horvath et al., 2000; Schueler et al.,
2001; Guy et al., 2003; Rudd and Willard, 2004]. This
organization within centromeres is also found in the great apes,
whereas lower primates only have monomeric a-satellites at their
centromeres [Thayer et al., 1981; Alves et al., 1994]. Human and
chimpanzee alphoid sequences underwent remarkably rapid
evolution in both species, mediated by massive interchromosomal
exchange and divergent intrachromosomal expansion leading to
the generation of species-specific a-satellite DNA [Archidiacono
et al., 1995; Warburton et al., 1996; Haaf and Willard, 1997].
Comparative analyses between humans and chimpanzees have
revealed that monomeric a-satellites have diverged less rapidly

than higher-order a-satellites [Rudd et al., 2006]. These findings
are remarkable since higher-order a-satellites are associated with
centromere function [Harrington et al., 1997; Ikeno et al., 1998;
Schueler et al., 2001; Spence et al., 2002] and might therefore be
expected to be subject to purifying selection. However, the more
rapid evolution of the higher-order a-satellites could have been
accompanied by rapidly coevolving centromere-associated pro-
teins. In support of this postulate is the rapid evolution of CENPC
proteins in plants and animals [Talbert et al., 2004]. Despite the
obvious functional significance of human centromeres, their highly
repetitive sequences have greatly hampered their inclusion in the
genome assembly [Eichler et al., 2004]. Only some chromosome
assemblies (including human chromosomes 2, 4, 6, 7, 8, 10, 11, 17,
18, X, and Y) contain considerable amounts of higher-order
a-satellite DNA [Rudd and Willard, 2004], and as yet none of the
human centromeres have been completely sequenced. Further, as
yet, none of the chimpanzee chromosome assemblies in the draft
sequence have reached higher-order a-satellites. More is known,
however, about the sequence structure and evolution of the
centromeric transition zone between the pericentromeric region
and the higher-order a-satellite arrays [Horvath et al., 2000, 2005;
She et al., 2004; Schueler et al., 2005]. For human chromosome 8,
a complete sequence contig is available that spans the entire
pericentromeric region [Nusbaum et al., 2006]. Especially well-
characterized in this respect is the pericentromeric region of the
human X chromosome. An age gradient is observed, with the
most distal a-satellites in the transition zones being ancient,
whereas more recently amplified repeats are located in the central
functional region. This pattern has also been observed in the
chimpanzee and other primates, implying that the primate X
centromere evolved through repeated expansion extending from
the central active region of centromeric a-satellites [Schueler
et al., 2001, 2005].
Comparative analyses of the pericentromeric regions in humans
and chimpanzees, as well as in other primates, have revealed
insights into their extraordinary dynamism, characterized by
extensive duplication, deletion, and rearrangement of large
segments of DNA [Eichler et al., 1996, 1997; Regnier et al.,
1997; Zimonjic et al., 1997; Orti et al., 1998; Jackson et al., 1999;
Bailey et al., 2001, 2002b; Horvath et al., 2000, 2003; Schueler
et al., 2001; Crosier et al., 2002; She et al., 2004]. The
pericentromeric regions of human chromosomes have been formed
via extensive duplications that have colonized pericentromeric
DNA (pericentromeric seeding). Secondary duplications of
larger mosaic blocks (pericentromeric swapping) occurred
subsequent to the seeding events, giving rise to a differential
distribution of these blocks among the great ape and human
pericentromeric regions [Jackson et al., 1999; Guy et al., 2000,
2003; Horvath et al., 2000; 2005; Locke et al., 2005]. The global
comparison of differences in the segmental duplication content of
human and chimpanzee genomes indicates that the human
genome has experienced more pericentromeric duplications than
the chimpanzee genome, suggesting that pericentromeric duplica-
tive expansion has been more pronounced in the human lineage
[Cheng et al., 2005].
GENE DUPLICATION AND OTHER
MECHANISMS RESPONSIBLE FOR CREATING
HUMAN-SPECIFIC GENES
Gene Duplications
As a consequence of the above-mentioned lineage-specific
segmental duplications, Cheng et al. [2005] demonstrated that at
HUMAN MUTATION 28(2), 99^130, 2007 115
least 180 genes and partial genes have become duplicated
specifically in the human genome, whereas at least 94 genes have
been duplicated specifically in the chimpanzee lineage. These
lineage-specific segmental duplications serve as a reservoir of
duplicated genes and it may be that some of these genes retained
their function and diverged such that they now confer hominid-
specific traits [Eichler, 2001; Bailey et al., 2002a,b; Samonte and
Eichler, 2002; Courseaux et al., 2003].
The potential of duplicated genes to evolve new functions by
sequence divergence was first mooted by Ohno [1970]. Released
from the functional constraints that operated upon their
parental source genes, duplicated gene copies are in principle free
to undergo neo-, sub-, or microfunctionalization [Hurles,
2004; Hancock, 2005]. Whereas neofunctionalization represents
the acquisition of a new function, subfunctionalization entails
the adoption of part of the function of the parental gene
by the duplicate copy [Hurles, 2004]. Microfunctionalization
describes the process by which duplicated genes come to encode
similar proteins with subtly different functions, such as olfactory
receptors [Hancock, 2005]. These processes are frequently
accompanied by divergence of spatial expression patterns between
human (and more generally eukaryotic) duplicated genes [Makova
and Li, 2003; Li et al., 2005]. However, most of the duplicates
become degraded [Nadeau and Sankoff, 1997; Li et al., 2001]; this
has consequently been termed a birth and death process [Nei
et al., 1997; Nei and Rooney, 2005]. Some of the duplicated genes
nevertheless adopt divergent expression profiles [Kim et al., 1989;
Gu et al., 1997; TomHon et al., 1997] and acquire novel functions
while evolving under different selective constraints and pressures
than the parental genes [Lynch and Conery, 2000; Prince and
Pickett, 2002; Kouprina et al., 2004b; Birtle et al., 2005; Yu et al.,
2006b; Svensson et al., 2006].
Several primate-specific genes and gene families are thought to
have arisen in this way, for example the melanin-concentrating
hormone-like genes [Courseaux and Nahon, 2001], the morpheus
gene family [Johnson et al., 2001], the chorionic gonadotropin
genes [Maston and Ruvolo, 2002], the SPANX genes of cancer/
testis-specific antigens [Kouprina et al., 2004b], the NBPF gene
family [Vandepoele et al., 2005], and many others [Samuelson
et al., 1990; reviewed in Gagneux and Varki, 2001; Zhang et al.,
2002a; Paulding et al., 2003]. The widespread existence of gene
families itself provides confirmation that gene duplication and
divergence has been of very considerable importance with respect
to the evolution of new biological functions. Quite frequently,
members of gene families with multiple paralogs are highly
rearranged in comparison with the source locus. These rearrange-
ments are mostly associated with domain or exon accretion, a
process that allows avoidance of potentially deleterious negative
gene dosage effects that might occur if two highly homologous
duplicate genes are maintained in the genome [reviewed by
Nahon, 2003; Babcock et al., 2003; Ciccarelli et al., 2005]. It can
be seen that the marked instability of segmental duplications
characterized by extensive genomic remodeling during
hominoid evolution has facilitated the generation of great
apespecific and human-specific chimeric genes [Eichler, 2001;
Nahon, 2003]. Furthermore, rearrangements in gene order during
the creation and/or the expansion of LCRs represent an important
mechanism of genome evolution, a phenomenon exemplified
by the DPY19L2 gene, which is located within LCRs [Carson
et al., 2006].
A genomewide survey of gene duplication across the great apes
and humans has been performed using cDNA arrays covering
29,619 different genes [Fortna et al., 2004]. Interestingly, humans
Human Mutation DOI 10.1002/humu
116 HUMAN MUTATION 28(2), 99^130, 2007
were found to possess a larger number of lineage-specific gene
duplications than the great apes. A total of 134 gene families were
identified that exhibited human lineage-specific gains, including
a number of genes with possible neuronal functions. Quite
remarkable is the positional bias of these gains since many were
found in the pericentromeric regions of chromosomes 1 and 9
[Fortna et al., 2004]. Importantly, the great majority of these copy
number gains between humans and chimpanzees as detected
by cDNA aCGH [Fortna et al., 2004] were also found in studies
using aCGH with genomic clones (BAC aCGH) [Cheng et al.,
2005; Wilson et al., 2006a; Goidts et al., 2006a]. These array-
based analyses have therefore served to widen the spectrum of
identified human-specific genes that have arisen since the
separation of human and chimpanzee lineages (e.g., the SMN2
gene) [Rochette et al., 2001], members of the olfactory receptor
gene [Trask et al., 1998], NANOG gene/pseudogene [Fairbanks
and Maughan, 2006], keratinocyte growth factor (KGF) [Zimonjic
et al., 1997], and immunoglobulin G receptor gene families (e.g.,
FCGR1) [Maresco et al., 1996, 1998], as well as 13 human
lineage-specific PRAME genes [Birtle et al., 2005].
A recent study of
9,600 gene families, identified by means of
a likelihood method that makes efficient use of comparative
genomic data [Hahn et al., 2005], examined gene birth and loss
among gene family members across five mammalian species
(human, chimpanzee, mouse, rat, and dog). By aligning 13,454
unambiguous orthologous genes of human and chimpanzee
genomes, a gain of 812 genes was specifically noted in the human
lineage, with 1,180 genes having been gained in the chimpanzee
lineage (J.P. Demuth and M.W. Hahn, personal communication).
Similarly, 332 genes were found to have been lost specifically in
the human lineage, with 1,043 genes having been lost in the
chimpanzee lineage. Thus, in total, the two primate genomes
were found to be distinguishable by the presence or absence
of some 3,731 genes (J.P. Demuth and M.W. Hahn, personal
communication).
Taken together, these findings confirm the view espoused by
Nahon [2003], that the concept of human-specific genes can no
longer be regarded as being heretical and that some of these genes
may well have been responsible for influencing the evolutionary
emergence of human-specific traits. Indeed, human-lineage
specific copy number expansions of genes important for brain
functions and/or speech development may have had a particularly
important impact on the human-specific evolution of these
processes [Fortna et al., 2004; Sikela, 2006].
Intragenic Duplications
Intragenic duplications may also contribute to the genomic
divergence between humans and chimpanzees. Thus, Jensen-
Seaman and Li [2003] have reported an intragenic duplication
involving the 60amino acid residue tandem repeats of the
chimpanzee semenogelin 1 gene, resulting in a protein nearly twice
as long as that encoded by the human SEMG1 gene. Semenogelins
1 and 2 represent the predominant structural proteins in human
semen and the different structure of semenogelin 1 in humans and
chimpanzee may be of significance in the context of the observed
differences in the consistency of semen between the two species.
Pseudogene Creation by Retroposition
Another mechanism of gene creation is the integration
of reverse-transcribed processed mRNAs into genomic DNA
mediated by the enzymatic machinery of L1 non-LTR retro-
transposons (retroposition) [Brosius, 1991; Long et al., 2003;
Human Mutation DOI 10.1002/humu
Pavlicek et al., 2006]. Approximately 70% of the
20,000 human
pseudogenes result from retroposition rather than genomic
duplication [Goncalves et al., 2000; Torrents et al., 2003].
Processed pseudogenes are usually intronless, lack regulatory
elements, and are frequently inactivated by microdeletions or
premature stop codons. There is, however, increasing evidence
that at least a proportion of them are functional and that
retroposition constitutes an important mechanism for the genera-
tion of new genes [reviewed in Brosius, 1999; Korneev et al., 1999;
reviewed by Betran and Long, 2002; Betran et al., 2002; Long
et al., 2003; Hirotsune et al., 2003; Strichman-Almashanu et al.,
2003; Emerson et al., 2004]. A high rate of retroposition occurred
in the primate and rodent lineages [Ohshima et al., 2003; Zhang
et al., 2004b], probably driven by the activity of transposable
elements such as LINEs and SVAs [Esnault et al., 2000; Torrents
et al., 2003; Zhang et al., 2003], which are able to transduce
non-transposon-derived DNA flanking their 30 ends to new
genomic locations, a process termed 30 transduction [Moran et al.,
1999; Goodier et al., 2000; Ostertag et al., 2003].
In a genomewide approach, Marques et al. [2005] estimated, on
the basis of a systematic analysis of selective signatures in
processed pseudogenes, that between 57 and 76 such retrogenes
emerged during primate evolution. The majority of these primate-
specific retrogenes are specifically expressed in the testis in
contrast to the widely expressed original source genes that they
were derived from. Therefore, several of these retrocopies could
have experienced a gain-of-function during spermatogenesis in
primates [Marques et al., 2005]. One such well-characterized
retrogene is glutamate dehydrogenase 2 (GLUD2), which
originated in the hominoid ancestor
23 Mya by retroposition
from the ubiquitously expressed glutamate dehydrogenase 1
(GLUD1) gene. The unique and brain-specific properties of this
enzyme, which plays a role in the brain metabolism of the key
neurotransmitter glutamate, are not intrinsic to the parental
GLUD1 gene but rather originated from amino acid substitutions
that occurred during a postduplication period of positive selection
during which brain size increased in the ancestors of both humans
and great apes [Burki and Kaessmann, 2004]. Thus, the GLUD2
gene represents a paradigm for the evolution of new functions
mediated by gene duplication during hominoid evolution.
Comparative analysis of human chromosome 22 and the
orthologous chromosome in the chimpanzee revealed a total
of six human-specific processed pseudogenes that probably
integrated into the human genome after the divergence of both
lineages [Watanabe et al., 2004]. The human/chimpanzee genome
comparison revealed the existence of 163 human-specific
retrotransposed gene copies and 246 that arose specifically in
the chimpanzee lineage [Mikkelsen et al., 2005]. From this finding
and the incomplete coverage of the chimpanzee genome, one
might infer that there may be in excess of 200 human lineage-
specific and 300 chimpanzee-specific retrocopies, some of which
could have subsequently acquired new functions. Moreover, as
suggested by Balakirev and Ayala [2003], although pseudogene
sequences are generally less well conserved than functional genes
and intergenic regions [Zheng et al., 2005], some pseudogenes
common to humans and other primates may nevertheless have
evolved under lineage-specific selective constraints and might
thus be associated with the evolution of human-specific
phenotypic traits. Among the variety of different functions
acquired by pseudogenes is the regulation of the mRNA stability
of the parental source gene, a phenomenon exemplified by the
makorin 1 (MKRN1) gene and its retrocopy [Hirotsune et al.,
2003]. Further comparative analysis of pseudogenes in the human

and chimpanzee genomes, including other primates as outgroups,
is required in order to ascertain the full spectrum of primate-
specific (and human lineage-specific) pseudogenes and their
functional significance.
Pseudogene-Mediated Gene Conversion
Pseudocopies not only represent a reservoir of genes that are
capable of developing new functions, they also have the potential
to mediate conversion of the parental source genes. It has been
proposed that pseudogene-mediated gene conversion may
contribute to adaptive evolution and, in so doing, potentiate an
adaptive peak shift [Cooper, 1999; Hansen et al., 2000]. In nevertheless clear that small inversions make a very significant
principle, the rapid evolution of a novel gene function could be
accomplished by the introduction of several substitutions simulta-
neously. An example of just such a conversion has recently been
identified by Hayakawa et al. [2005], who noted that the
SIGLECP16 pseudogene has converted its functional counterpart,
SIGLEC11. This conversion occurred specifically in the human
lineage rather than in that of the chimpanzee. The converted
region includes a 50 upstream region and exons encoding the sialic
acid recognition domain. Strong Siglec-11 expression was observed
in human cortex microglia (in contrast to chimpanzee and
orangutan), possibly due to changes in 50 regulatory sequences.
Gene conversions do not seem to be rare events. Indeed, by
analyzing 2,641 duplicated genes in mouse and rat, 18% were
found to exhibit signs of gene conversion [Ezawa et al., 2006].
Pathological changes in functional genes mediated by gene
conversion through a pseudogene are not uncommon [Chiu
et al., 1997; Gupta et al., 2005; reviewed by Cooper, 1999;
Balakirev and Ayala, 2003]. Detailed analyses of gene conversion
events involving duplicated genes in humans and chimpanzees
should provide information as to how frequently human lineage-
specific gene conversion has contributed to human gene evolution.
STRUCTURAL DIVERGENCE MEDIATED
BY INVERSIONS
As outlined above, genomic duplications as well as lineage-
specific deletions have contributed significantly to genome
evolution in great apes and humans. In addition, submicroscopic
inversions represent structural variants of the human and
chimpanzee genomes [Fortna et al., 2004; Cheng et al., 2005;
Feuk et al., 2005; Hughes et al., 2005; Newman et al., 2005;
Szamalek et al., 2006c]. Thus, for example, the Y chromosomes of
humans and chimpanzees differ by two inversions, one in the
chimpanzee lineage spanning 5 Mb, the other, of length 1.5 Mb,
being specific to humans [Hughes et al., 2005].
In a genomewide human/chimpanzee comparison, a total
of 174 putative inversions were identified by Newman et al.
[2005]. Their comparative approach included the mapping of

1.8 million fosmid end sequences from a genomic library of a
single chimpanzee (10-fold physical coverage of the chimpanzee
genome) against the human genome reference sequence. A total
of 15 of these rearrangements spanned more than 20 Mb and
correspond to the cytogenetically visible large pericentric inver-
sions. The remaining 159 putative inversions of 41.5 kb clearly
contribute to the virtually uncharacterized bulk of submicroscopic
structural variation between the human and chimpanzee genomes.
In contrast to this fosmid mapping strategy, Feuk et al. [2005]
performed direct alignments of the chimpanzee draft sequence
against the human reference sequence. These comparisons
disclosed 1,576 inversions ranging in size from 23 bp to 62 Mb,
with the largest representing the known karyotypically visible
HUMAN MUTATION 28(2), 99^130, 2007 117
pericentric inversions of chromosomes 4, 5, 15, and 18. Employing
gene order comparisons for the human and chimpanzee genomes,
Szamalek et al. [2006c] succeeded in identifying 71 submicro-
scopic inversions. Nine of these that contained more than three
genes were then experimentally validated and the authenticity
of five of the nine inversions, ranging in size from
800 kb to

4.4 Mb, was confirmed. There is some overlap between the
inversions identified in the above three studies. However, owing to
experimental limitations inherent to all three studies, the precise
number of inversions that distinguish the human and chimpanzee
genomes is difficult to ascertain with any degree of accuracy. It is
contribution to the structural divergence between the two
genomes. Since the great majority of the inverted regions contain
genes and in some cases are even associated with the disruption
of genes at the inversion breakpoints [Newman et al., 2005],
their potential to cause lineage-specific gene loss and differences
in expression through position effects would appear to be quite
considerable.
The breakpoints of many of the identified inversions map to
sites of segmental duplications, an observation already made in the
context of deletions and duplications. This serves to underline
the key role that segmental duplications play as mediators of
rearrangements in hominoid genomes due to the intrinsic
propensity of these regions to recombine [Feuk et al., 2005;
Newman et al., 2005; Szamalek et al., 2006c]. To assess the
significance of these inversions for the evolution of humans and
chimpanzees it is important to study the lineage specificity of the
different inversions. This is necessary because some of the
inversions identified as being divergent sites between human and
chimpanzee actually represent intraspecies polymorphisms [Feuk
et al., 2005; Szamalek et al., 2006c].
STRUCTURAL DIVERGENCE (INTERSPECIES
VARIABILITY) AND DIVERSITY
(INTRASPECIES VARIANCE)
Inversions
Of the 23 verified submicroscopic inversions described by Feuk
et al. [2005], three (1, 17, and 700 kb in size, respectively) turned
out to be polymorphic variants in humans. The 700-kb inversion
located on chromosome 7 was also identified by Szamalek et al.
[2006c], together with four inversions of 1 to 4.4 Mb that were
polymorphic in the chimpanzee. These findings indicate that both
the structural divergence and the structural diversity found in
humans and chimpanzees are rather greater than was initially
expected [Carter, 2004; Buckley et al., 2005; Feuk et al., 2006].
Inversions contribute to the unexpectedly high degree of
structural variation in the human genome. In the study of Tuzun
et al. [2005], 56 inversions were identified that presumably
represent regions of human polymorphism. Structural diversity
between humans is therefore more the rule rather than the
exception. Prior to these studies, some variant inversions or gene
duplications were already known and may turn out to be important
in a clinical genetics context (summarized in Supplementary
Table S4). An inversion variant in the Williams-Beuren syndrome
(WBS) region in 7q11.23 has been found in 25 to 33% of
transmitting parents of individuals with WBS, implying that the
inversion could predispose this region to the WBS deletion
[Osborne et al., 2001; Scherer et al., 2005]. Further, submicro-
scopic inversions of 15q11q13 have been detected at dispropor-
tionately high frequency (67%) in the mothers of Angelman
syndrome patients with class II (BP2/3) deletions [Gimelli et al.,
Human Mutation DOI 10.1002/humu
118 HUMAN MUTATION 28(2), 99^130, 2007
2003], as compared to 9% in the general population. Only
recently, a heterozygous inversion encompassing the NSD1 gene
at 5q35.3 has been identified in fathers of children with
Sotos syndrome [Visser et al., 2005]. Since the latter inversion
polymorphism is very common in the general population, further
work will be required to confirm or exclude its involvement in
predisposition to Sotos syndrome [Visser et al., 2005].
De novo recurrent chromosome rearrangements, involving
human chromosomes 4 and 8, are the recombinant products of
heterozygous inversions present in the transmitting parent.
Furthermore, these inversions on 4p16 and 8p23 have been
detected in 12.5 and 26% of control subjects, respectively, and
thus represent quite frequent polymorphisms [Giglio et al., 2001,
2002]. As with the inversions involving 5q35.3, 7q11.23, and
15q11q13, these inversions are flanked by paralogous segmental
duplications (SD), a finding which emphasizes the importance of
duplicated regions as actual and potential mediators of inversions.
Duplications and Deletions
Copy number variations (CNVs; the accepted term for
polymorphic duplications and deletions) would appear to be
responsible for millions of nucleotides of heterogeneity between
extant human genomes. Indeed, it is likely to contribute to a much
larger extent to human diversity, and probably also to disease
susceptibility, than has previously been assumed [Carter, 2004;
Buckley et al., 2005; Feuk et al., 2006; Wilson et al., 2006b].
DNA microarray analysis has proved to be a powerful tool for
the detection of CNVs 44050 kb in size [Iafrate et al., 2004;
Sebat et al., 2004; Sharp et al., 2005; Goidts et al., 2006]. Thus,
extrapolating to the entire genome, any two human individuals
are likely to be distinguishable by approximately 100 CNVs 440
50 kb [Feuk et al., 2006]. CNVs were also detected through the
alignment of paired-end sequence data from a human fosmid
genomic library against the human reference sequence [Tuzun
et al., 2005]. In this study, 102 regions of the human genome were
identified that constitute sites of polymorphic deletion of 48 kb in
length. Human CNVs are significantly overrepresented in the
proximity of telomeres and centromeres, and in simple tandem
repeat sequences [Tuzun et al., 2005]. Furthermore, human CNVs
are nonrandomly distributed, with a four- to 12-fold greater
frequency of occurrence close to SDs [Sharp et al., 2005; Tuzun
et al., 2005; Goidts et al., 2006; Kriek et al., 2006]. In chimpanzee
populations, a 20-fold enrichment of CNVs at sites of SDs has
been noted [Perry et al., 2006]. This indicates that SDs have an
increased tendency to vary in copy number, thereby mediating
common human (and chimpanzee) genetic variation [Mehan
et al., 2004; Perry et al., 2006]. Further, CNVs are enriched in
protein-coding genes that have experienced significantly elevated
synonymous and nonsynonymous nucleotide substitution rates as
compared to orthologous genes in the mouse. The elevated coding
sequence divergence suggests that a subset of genes within CNVs
may be associated with an adaptive increase in gene dosage
[Nguyen et al., 2006b].
SNP genotyping and oligonucleotide microarray analyses of
long-range PCR products have revealed that polymorphic dele-
tions ranging from 0.510.5 kb are widespread in the human
genome [Conrad et al., 2006; Hinds et al., 2006; McCarroll et al.,
2006]; indeed, by these means,
1,000 regions harboring such
deletion polymorphisms were identified in the human genome.
This number is, however, likely to represent a conservative
estimate of the total number of polymorphic deletions in the
human genome since the overlap of deletion variants identified in
Human Mutation DOI 10.1002/humu
these studies is rather small. We may surmise that the methods
applied do not systematically detect all common polymorphic
deletions and that the actual number may eventually prove to be
considerably higher [Eichler, 2006].
Structural Variation and Disease Susceptibility
Structural variants such as inversions may impart disease
susceptibility to the offspring of heterozygote inversion carriers
by virtue of their potential to give rise to additional genomic
rearrangements. Copy number variations can also be associated
with disease susceptibility as has been exemplified by the CC
chemokine ligand 3-like 1 (CCL3L1) gene, located within a site
of segmental duplication. Copy number variations of CCL3L1-
containing duplicons have been identified in different human
populations and a CCL3L1 copy number that is lower than
the population average is associated with enhanced susceptibility
to human immunodeficiency virus (HIV)/AIDS [Gonzalez
et al., 2005]. Copy number variation of human FCRG3 genes
also appears to determine susceptibility to immunologically
mediated glomerulonephritis [Aitman et al., 2006].
Leaving aside the potential pathological significance of some
structural variants, it may be that other structural variants have
been advantageous during human evolution. If a structural
polymorphism such as an inversion has been maintained in a
population for a long period of time, a selective advantage accruing
to carriers may be inferred. In contrast to SNPs, heterozygous
inversions are more likely to be naturally eliminated from the
population since crossing over within the inverted segments results
in genetically unbalanced offspring. If, however, such inversion
heterozygosity is maintained over an extended period of time, a
selective advantage resulting from this heterozygosity might be
assumed. A human polymorphic inversion that appears to have
evolved under positive selection has recently been reported by
Stefansson et al. [2005]: a 900-kb inversion polymorphism at
17q21.31 within a region that includes the microtubule-associated
protein tau (MAPT) gene. Chromosomes with the inverted
segment in different orientations represent two distinct lineages,
termed H1 and H2, that appear to have diverged for as many as
3 million years with no evidence of recombination between them.
The high frequency of H2 chromosomes in European populations
is consistent with the rapid expansion of this allele under the
influence of positive selection in Europe and the Near East
[Stefansson et al., 2005].
The detailed analyses of these polymorphic variants with regard
to structure, segregation and frequency in the human population
promises to lead to significant new insights into the nature and
impact of both negative and positive selection pressure during
human evolution. Whether with regard to studies of human
evolution or disease susceptibility, the analysis of human genome
diversity, and in particular those changes involving structural
variation, will benefit enormously from comparisons with the
genomes of other primates. These comparisons should help to
reveal ancestral states, lineage-specific evolutionary trends, and
signatures of adaptive diversity, as well as their phenotypic
functional consequences.
OVERVIEW
The plethora of genetic changes that differentiate humans from
their closest relatives the chimpanzees affect multiple levels and
are much more complex than was assumed prior to the advent
of genomewide comparative analyses. A considerable number of
human genes have so far been identified that evolved under recent

positive selection. Some of these may turn out to have had a
major impact on the evolution of human-specific traits.
Further, differential expression patterns of large sets of genes can
discriminate the transcriptomes of humans and chimpanzees.
Even more striking is that transcriptomes and proteomes appear
to have coevolved in humans, implying a complex interplay of
evolutionary forces acting at different levels of the expression
pathway.
The continuing search for differences between the human and
chimpanzee genomes has identified extensive structural variation
that has been frequently mediated by recombination-prone
segmentally duplicated sequences and is likely to be associated
with a multitude of functional changes that still largely remain to
be identified. The identification of the individual components
of the structural divergence between the human and chimpanzee
genomes has been accompanied by the discovery of very
considerable lineage-specific structural diversity. Indeed, copy
number polymorphisms as well as inversions have turned out
to be a major contributor to the polymorphism manifest in
extant human genomes. At least some of these structural variants
are likely to turn out to be associated with disease susceptibility
or complex trait development. The parallel investigation
of the corresponding regions in the chimpanzee and other primate
genomes will not only help us to distinguish between
genetic divergence and diversity, but is also likely to provide
important new insights into the evolutionary history of our
extended primate family.
ACKNOWLEDGMENTS
We thank Violaine Goidts, Justyna M. Szamalek, Catharina
Sandig, Werner Schempp, Stefan Mu ller, and Matthias Platzer
for their scientific contributions to the ongoing work in Ulm,
and Matthew Hahn for access to data prior to publication. We
also thank Horst Hameister and James M. Sikela for helpful
discussions.
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