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Large-Scale Variation Among Human and Great Ape Genomes Determined by Array Comparative Genomic Hybridization1Department of Genetics, Center for Computational Genomics and Center for Human Genetics, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA; 2Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94143, USA;3 Dipartimento di Anatomia Patologica e di Genetica, Sezione di Genetica, University of Bari, Bari, Italy
Large-scale genomic rearrangements are a major force of evolutionary change and the ascertainment of such events between the human and great ape genomes is fundamental to a complete understanding of the genetic history and evolution of our species. Here, we present the results of an evolutionary analysis utilizing array comparative genomic hybridization (array CGH), measuring copy-number gains and losses among these species. Using an array of 2460 human bacterial artificial chromosomes (BACs) (12% of the genome), we identified a total of 63 sites of putative DNA copy-number variation between humans and the great apes (chimpanzee, bonobo, gorilla, and orangutan). Detailed molecular characterization of a subset of these sites confirmed rearrangements ranging from 40 to at least 175 kb in size. Surprisingly, the majority of variant sites differentiating great ape and human genomes were found within interstitial euchromatin. These data suggest that such large-scale events are not restricted solely to subtelomeric or pericentromeric regions, but also occur within genic regions. In addition, 5/9 of the verified variant sites localized to areas of intrachromosomal segmental duplication within the human genome. On the basis of the frequency of duplication in humans, this represents a 14-fold positional bias. In contrast to previous cytogenetic and comparative mapping studies, these results indicate extensive local repatterning of hominoid chromosomes in euchromatic regions through a duplication-driven mechanism of genome evolution. [Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: O. Ryder, L. Faust, and E. Adams.]
The evolution of human and nonhuman primate genomes has been studied at two levels, karyotypic differences and single-base-pair nucleotide differences (Yunis et al. 1980  ; Yunis
and Prakash 1982; Muller et al. 1989; Kaessmann et al. 2001; Muller and
Weinberg 2001). To date, no ascertainment has been made of the
frequency, extent, or distribution of large-scale sequence gain or loss
events, defined here as copy-number altering events involving >10 kb
of sequence, yet events that are undetectable at the cytogenetic level.
Events of this size have the potential to significantly impact the gene
complement and genome structure of closely related species; however,
genome-wide experimental comparative analyses at this scale have been
impossible due to the lack of efficient methods of comparing genomes
with accuracy and a high level of resolution. With the advent of the
human genome reference sequence, however, new approaches have emerged
that facilitate the simultaneous detection and high-resolution mapping
of large-scale DNA variation across the entire genome (Snijders et al.
2001). One such method, array comparative genomic hybridization (array
CGH), has demonstrated the ability to reliably detect DNA copy-number
changes between genomic DNA samples with the resolution of a single BAC
clone (Pinkel et al. 1998; Albertson et al. 2000; Snijders et al.
2001).
Our array CGH procedure involves the hybridization of differentially
labeled specimen and reference genomic DNA to an array of large-insert
genomic clones. The hybridization intensity ratio at each array locus
is proportional to the copy-number ratio between genomic DNA samples,
which is used as a measure of putative regional gains and losses. The
microarray used in this study consisted of 2460 large-insert human BAC
clones that had been mapped previously by STS content and validated by
FISH (Snijders et al. 2001
Large-scale sequence duplication and deletion events have the potential
to significantly impact the structure and evolution of the primate
genome. At the simplest level, sequence gain and loss events can alter
the gene complement of an organism, which may result in phenotypic
variation susceptible to selection pressures. Gene loss has been
proposed as an important force in driving the evolution of eukaryotic
genomes in response to selective pressures such as altered growth
conditions in yeast, or infectious disease in the human population
(Olson 1999 Using normal human genomic DNA as the reference, we compared relative hybridization intensities among four great ape species, chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus), in pairwise comparisons with human. Multiple individuals of each species were examined in independent experiments, and array loci were scored as potential variants only if a consistent increase or decrease in hybridization intensity ratio was observed across all trials and all individuals of each species (see Methods). This approach ensured the detection of fixed differences between the species, but eliminated potential large-scale polymorphisms within species for further analysis. The application of such conservative criteria, however, was essential to identify sites of interspecific copy number differences as opposed to spurious artefacts. We examined the distribution and the sequence context of these sites to provide some insight into the significance of such variation as a potential force underlying chromosomal change among humans and nonhuman primates. This study, therefore, presents the first genome-wide comparison of great ape species, with the level of resolution afforded by array CGH.
Genomic DNA from multiple unrelated individuals of each primate species were analyzed using array CGH, and only sites displaying copy-number variation in all individuals were scored as positive (Fig. 1). Four great ape species were examined in this study, common chimpanzee (n = 2), pygmy chimpanzee (n = 4), gorilla (n = 5), and orangutan (n = 4). In addition, several hybridizations with individual primate genomic DNA samples were repeated and the results compared with previous hybridizations to test the consistency of the hybridization results. On the basis of these criteria, 63 sites of variant intensity ratios (38 reductions and 25 increases) were consistently identified in primate–human comparisons and mapped to the human reference genome sequence (Fig. 1; Supplemental Table 1). Both lineage-specific and shared-ratio differences were observed among the great apes. Lineage-specific differences, however, predominated (Fig. 2B). As expected, the quantity of variant sites detected in each great ape species was in proportion to the estimated divergence times of each species (Goodman 1999 ), as orangutan showed the greatest
number of ratio differences and the chimpanzee species demonstrated the
fewest ratio differences.
We chose a subset of the 63 putative variant sites for detailed experimental validation and verification of the array CGH approach. Seven sites with increased primate signal intensity ratios, therefore potential duplications with respect to the human genome, were assessed by interphase and metaphase FISH. In all cases, a genomic duplication was detected among the great apes when probing with the human single-copy arrayed BAC. Among these variants, we observed both intra- and interchromosomal duplications, duplications within homologous chromosomes and between nonhomologous chromosomes, respectively, which were readily resolved at the metaphase and interphase levels (Fig. 3A–C). Similarly, seven sites with reduced primate signal intensity ratios, potential interspecific deletions, were examined by FISH. We encountered one instance in which there was a complete absence of signal, suggesting that a deletion of at least the size of the arrayed BAC (175 kb by PFGE, data not shown) had occurred (Table 1; Fig. 3A). The extent of this deletion was subsequently confirmed by STS-content mapping and Southern analysis (data not shown). Reduced FISH signal intensity was observed for several of the remaining six sites tested, yet deletions could not be demonstrated convincingly by comparative FISH. Considering that the Log2 ratios for these unverified sites were decreased to a lesser extent than the complete deletion demonstrated in orangutan by RP11-171I8, we believe these sites were partial deletions of the arrayed BAC sequence. Thus, we sought an alternative approach to validate these potential partial deletions.
We developed a BAC end sequence-based strategy to verify the potential partial deletions that were not confirmed by comparative FISH. This strategy compared the insert size of primate BACs linked to a variant site with the estimated BAC insert size according to the human genome reference sequence (Methods; Fig. 4B). A disparity between the experimentally determined primate BAC insert size, and the estimated insert size on the basis of sequence similarity searches against the human genome sequence, was indicative that a partial deletion had occurred. For a putative deletion in chimpanzee 11q12, this approach identified a  38-kb disparity between chimpanzee BAC insert size and
the human equivalent insert size (Fig. 3B). Examination of the
underlying human sequence revealed the presence of a 40-kb tandem
intrachromosomal duplication event that contained a partial duplication
of the BC005998 gene. Subsequent Southern analysis confirmed the
deletion of the proximal BC005998 duplicon in the chimpanzee genome
(Fig. 3B). Thus, we had successfully identified a deletion in the
chimpanzee genome using array CGH that was below the level of
cytogenetic detection and involved the partial deletion of a BAC clone
on the array. We believe that the tandem duplication was present prior
to the divergence of the great ape lineages, as the gorilla and
orangutan did not show reduced intensity ratios at this locus. Two
other predicted sites were investigated for possible deletions by this
method; however, no size discrepancies were detected by this BAC
end-mapping approach.
Combined, the experimental validation results suggest that array CGH offers excellent sensitivity in detecting genomic duplications, as 100% of array CGH-detected variant sites were verified experimentally by comparative FISH; however, deletions were not detected with similar efficiency, as only two deletions have been verified experimentally. Unlike intensity ratio increases, the intensity ratio decreases reported by array CGH may be due to a number of other factors, such as extensive sequence divergence or dramatic restructuring of loci as a result of repeat-content variation. Therefore, sites of reduced intensity ratios, without significant size differentials between species by our BAC end-sequence-based approach, remain important targets for further comparative sequence analysis. As large-insert genomic libraries become available from a number of the great ape species (Eichler and DeJong 2002 ), high-quality BAC sequencing of such
regions will provide the most efficient method for assessing the
molecular nature of these differences.
Previous studies have suggested that the majority of fixed large-scale variation between the genomes of humans and great apes localize to gene-poor heterchromatic regions (Verma and Luke 1991 ;
Archidiacono et al. 1995; Trask et al. 1998). In light of these
predictions, we examined the genomic landscape around the nine
experimentally verified duplications (n = 7) and deletions
(n = 2). Gene content within the vicinity of a bonafide
genomic rearrangement site was assessed by considering the proximal and
distal 100 kb flanking the array BAC-linked STS, or by placing the BAC
end sequences for the variant clone against the human genome assembly
by sequence similarity searches (Methods). Overall, we found that six
of nine experimentally verified sites of rearrangement lie within or in
close proximity to actively transcribed euchromatic regions (Table 1).
These results suggest that gene-rich regions are susceptible to
copy-number changes between humans and great apes, and that array CGH
can effectively detect such rearrangements. Such regional differences
in copy number could, in theory, underlie gene-expression differences
that have been predicted and observed between humans and great apes
(King and Wilson 1975; Enard et al. 2002).
The distribution of sites along the interstitial euchromatic regions of
human chromosomes, determined by viewing the BAC-linked STS on the
human assembly, shows that these events show no bias toward the
subtelomeric and pericentromeric regions of human chromosomes. A total
of 9 of the 63 variant sites occurred within 2 Mb of a subtelomeric
region or pericentromeric region, and the remaining 86% of sites
(54/63) mapped to euchromatic regions of the human genome. This result
demonstrates that the large genomic insertion and deletion events
occurred in euchromatic, and potentially gene-rich regions, and were
not limited to the heterochromatic expansions observed in previous
karyotypic analyses (Yunis et al. 1980
During our analysis of potential large-scale variation, we noticed that
certain chromosomes showed a disproportionately large number of variant
sites (Fig. 2A). Interestingly, many of the same chromosomes
enriched for rearrangements (chromosomes 4, 7, 8, 16, 17, and 22) have
also been shown to be enriched for segmental duplications (Bailey et
al. 2002
To more specifically test this association, we examined the sequence
context for the nine verified structural rearrangements detected in
this study. Segmental duplications comprise an estimated 5% of the
total human genome sequence, yet we found 5/9 (56%) of our validated
rearrangements were within close proximity to segmental duplications,
an 11-fold bias (Bailey et al. 2002 In summary, our results show that array CGH technology is a powerful approach for interrogating large-scale differences among the genomes of closely related species. It can successfully identify large-scale deletions and duplications too small to be detected by standard karyotype analysis, yet too large to be readily resolved by whole-genome shotgun sequencing approaches. Our analysis of the orangutan genome using human BAC microarrays suggests that 3% sequence divergence is sufficient for cross-species comparisons by this method. Array CGH is therefore a valuable first step to target regions for specialized study—and is therefore amenable to many cross-species comparisons in which entire genomes are unlikely to be sequenced. Second, our analysis of primate genomes indicates that large-scale events are not uncommon within genic regions. Whereas large-scale differences among great apes have been documented within heterochromatic regions such as pericentromeric DNA, this is among the first demonstrations of large-scale differences within euchromatic DNA. We have characterized deletions and duplications ranging in size from 40–175 kb and have provided a first approximation the frequency of such events at a genome-wide level. Rate estimation of such events will require more uniform representation of the genome as well as resolution of regions enriched for segmental duplications. Perhaps the most striking finding is that the events are nonrandomly distributed. Regions within or in the vicinity of segmental duplications show a proclivity to delete and duplicate. Whereas such genomic variation has commonly been reported in association with de novo rearrangements associated with disease, these data implicate a homology-driven mechanism for the generation of fixed differences that distinguish closely related species. The biological significance of these events and their relationship to gene expression variation among primates await further characterization.
Human BAC Arrays Arrays were prepared as described by Snijders et al. (2001) .
Briefly, ligation-mediated PCR was used to prepare DNA representations
of 2460 human BAC and P1 artificial chromosome (PAC)
clones. The DNA was suspended in 20% DMSO and spotted in triplicate
onto chromium-coated microscope slides using a custom-built printer
with capillary printing pins. The entire array of 7500 spots filled
a 12-mm x 12-mm square. Each clone on the array contained at least
one STS mapped to the human reference assembly sequence, allowing the
underlying genomic sequence to be assessed. All clones on the array
were cytogenetically mapped by FISH, confirming 93.4% as single copy.
In addition, extensive analyses of cell lines containing known
single-copy aberrations were performed to allow recognition of clones
that contained significant amounts of sequence that mapped to multiple
sites in the human genome. Data from such clones were excluded from the
analysis presented here. On average, the array provides a resolution of
one genomic clone for every 1.4 Mb of human genomic sequence. It should
be noted that by performing this analysis with a human BAC array,
deletions that have occurred specifically within the human lineage with
respect to other primate genomes cannot be readily detected. Deletions
in the human lineage with respect to the chimpanzee may become apparent
as the chimpanzee genome project progresses, and sites detected via
this method will provide landmarks for where the chimpanzee and human
sequence maps may vary significantly. For the other primate species for
which no dedicated genome project is anticipated, it may be necessary
to develop reciprocal primate BAC arrays to detect large-scale
deletions that have occurred within the human genome.
Primate DNA Samples
Array Comparative Genomic Hybridization The ratio variation among clones at constant copy number (most of the clones on the array) was significantly higher for great ape/human comparisons than for human/human comparisons. This ratio variation complicated recognition of interspecies copy-number differences that might be affecting only a portion of a BAC, and would therefore show a ratio change of reduced magnitude compared with the expected value for single- or multi-copy changes. Fixed copy-number differences between species should result in no copies in the genome for a deletion event, or four copies for a duplication event. Thus, if the change affected the entire BAC, one should see Log2 ratios of minus infinity or 1 for deletions and duplications, respectively. However, several factors modify this expectation, including the following: (1) changes that affect only part of the BAC, which are predicted to produce less dramatic ratio differences, (2) whether or not the arrayed BAC contains duplicated sequences, and (3) incomplete suppression of the repetitive sequences in the great ape genome by human Cot-1 DNA.
Fluorescence in Situ Hybridization
BAC Analysis
We thank Oliver Ryder, Lisa Faust, and Erin Adams for providing primate material for this study. This work was supported, in part, by NIH grants GM58815 and HD043569 and U.S. Department of Energy grant ER62862 to E.E.E., NCI grants CA83040 to D.P. and CA84118 to D.A., and the financial support of Telethon, CEGBA (Centro di Eccellenza Geni in campo Biosanitario e Agroalimentare, and MIUR (Ministero Italiano della Istruzione e della Ricerca) to N.A. The financial support of the W.M. Keck Foundation, Vysis Inc., and a grant from the Charles B. Wang Foundation to the Center for Computational Genomics, Case Western Reserve University are also gratefully acknowledged. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Corresponding author.  E-MAIL eee{at}cwru.edu; FAX (216) 368-3432. Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1003303.
 /non- satellite junction at 16p11. Hum. Mol. Genet. 9: 113-123.
Received November 18, 2002; accepted in revised format January 11, 2003. 13:347-357 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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