- Research article
- Open Access
Evolutionary analysis of the highly dynamic CHEK2duplicon in anthropoids
© Münch et al; licensee BioMed Central Ltd. 2008
- Received: 06 May 2008
- Accepted: 02 October 2008
- Published: 02 October 2008
Segmental duplications (SDs) are euchromatic portions of genomic DNA (≥ 1 kb) that occur at more than one site within the genome, and typically share a high level of sequence identity (>90%). Approximately 5% of the human genome is composed of such duplicated sequences. Here we report the detailed investigation of CHEK2 duplications. CHEK2 is a multiorgan cancer susceptibility gene encoding a cell cycle checkpoint kinase acting in the DNA-damage response signalling pathway. The continuous presence of the CHEK2 gene in all eukaryotes and its important role in maintaining genome stability prompted us to investigate the duplicative evolution and phylogeny of CHEK2 and its paralogs during anthropoid evolution.
To study CHEK2 duplicon evolution in anthropoids we applied a combination of comparative FISH and in silico analyses. Our comparative FISH results with a CHEK2 fosmid probe revealed the single-copy status of CHEK2 in New World monkeys, Old World monkeys and gibbons. Whereas a single CHEK2 duplication was detected in orangutan, a multi-site signal pattern indicated a burst of duplication in African great apes and human. Phylogenetic analysis of paralogous and ancestral CHEK2 sequences in human, chimpanzee and rhesus macaque confirmed this burst of duplication, which occurred after the radiation of orangutan and African great apes. In addition, we used inter-species quantitative PCR to determine CHEK2 copy numbers. An amplification of CHEK2 was detected in African great apes and the highest CHEK2 copy number of all analysed species was observed in the human genome. Furthermore, we detected variation in CHEK2 copy numbers within the analysed set of human samples.
Our detailed analysis revealed the highly dynamic nature of CHEK2 duplication during anthropoid evolution. We determined a burst of CHEK2 duplication after the radiation of orangutan and African great apes and identified the highest CHEK2 copy number in human. In conclusion, our analysis of CHEK2 duplicon evolution revealed that SDs contribute to inter-species variation. Furthermore, our qPCR analysis led us to presume CHEK2 copy number variation in human, and molecular diagnostics of the cancer susceptibility gene CHEK2 inside the duplicated region might be hampered by the individual-specific set of duplicons.
- World Monkey
- Pericentromeric Region
- CHEK2 Gene
- Rhesus Macaque Genome
- Chimpanzee Chromosome
Segmental duplications (SDs) are euchromatic portions of genomic DNA (≥ 1 kb) that occur at more than one site within the genome and typically share a high level of sequence identity (>90%) . Both in situ hybridization and in silico analyses have shown that ~5% of the human genome is composed of duplicated sequences [2–4]. Duplications that can be traced to an ancestral or donor location are named duplicons. Based on a neutral model of genome evolution , duplicons with approximately 10% sequence divergence correspond to duplication events that have occurred 30–40 million years ago (MYA), i.e. before the radiation of Old World monkey and hominoid species . Furthermore, a conspicuous bias of interchromosomal SDs toward pericentromeric regions, or euchromatin/heterochromatin transition regions in general, was detected [7, 8]. Fine-scale analyses of pericentromeric regions disclosed a two-step model for the formation of such dynamic regions. An initial pericentromeric "seeding" event followed by subsequent exchange ("swapping") of duplicon blocks between pericentromeric regions has been proposed [9, 10]. Both homologous and non-homologous processes were shown to be involved in "seeding" and "swapping" of pericentromeric SDs in human and great ape genomes [9–13] (for review see ). The duplicative architecture of human and higher primate genomes has been shown to be a major force promoting rapid evolutionary turnover . Although the predisposition to expansion of interspersed segmental duplication is common to human and great apes, it appears that many species-specific duplication events have taken place at different regions of their genomes. Interestingly, two independent approaches determined the fraction of species-specific SDs in chimpanzee and human to be ~30%, while ~66% of SDs seem to be shared between both species [16, 17]. Thus, species-specific SDs are thought to have contributed to a larger extent to the genetic difference between chimpanzee and human than single-base mutations . Moreover, SDs seem to be sites of recurrent large-scale structural variations [18–20] and it has been estimated that ~20% of SDs are polymorphic within the human and chimpanzee genome .
Interestingly, among all human chromosomes the Y chromosome has the highest relative SD content [2, 6, 16, 21–23]. Recently, we have analyzed 866 kb of Y-chromosomal non-palindromic SDs delineating the four euchromatin/heterochromatin transition regions in Yp11.2/Yp11.1, Yq11.1/Yq11.21, Yq11.23/Yq12 and Yq12/YPAR2 [24, 25].
Here we report the detailed investigation of the CHEK2 duplicons, one of which is embedded within the analyzed Yq11.1/Yq11.21 SD cluster. The ancestral duplicated region, containing the proximal part of the functional CHEK2 gene (CHK2 checkpoint homolog S.pombe) and the distal part of TTC28 (tetratricopeptide repeat domain 28), is located in 22q12.1 [10, 25–27]. CHEK2 has been shown to be a multiorgan cancer susceptibility gene . Interestingly, CHK2, the protein encoded by CHEK2, is a cell cycle checkpoint kinase acting in the DNA-damage response signalling pathway . Cell cycle checkpoints monitor the structural integrity of chromosomes before their progression through crucial cell cycle stages. CHK2 homologues were found in yeast and higher eukaryotes [29–33] indicating an important role throughout eukaryotic evolution in controlling the integrity of the genome. The continuous presence of the CHEK2 gene in all eukaryotes  and its important role in maintaining genome stability  prompted us to investigate the duplicative evolution and phylogeny of CHEK2 and its paralogs during anthropoid evolution. We applied a combination of comparative FISH and in silico analyses. In addition, we used inter-species quantitative PCR for further validation and for detection of intra-species specific CHEK2 copy number variations.
Y-chromosomal cosmid library screening
We screened the LLOYNC03 "M" (Lawrence Livermore National Laboratory) Y-chromosomal library (5.5xY chromosome coverage) for CHEK2 duplicon containing cosmids using a Y-chromosomal probe generated by the following PCR-conditions: 95°C for 5 min, 40 cycles of 94°C for 30 sec, 62°C for 30 sec and 72°C for 1 min and finally 72°C for 5 min. The following primers were used to generate a 575 bp probe: IP-cos-SD1-for 5'-ACCCCCTTAGTAGCGTCCTTAGCTC-3' and IP-cos-SD1-rev 5'-ACCACCGGAGTTTCACAAAGAAAGT-3'. The gel-purified probe was radioactively labelled according to the 'random priming' protocol . Prehybridization and hybridization of the high density gridded filters were carried out for 18 hrs at 42°C according to the manufacturer's protocol. Final filter-washing was carried out in 1%SDS/2xSSC solution for 1 hr at 65°C. Primers located proximal and distal of the derivative Y-chromosomal CHEK2 duplicon were used for PCR (conditions see above) to identify cosmids containing the entire duplicon:
PB-cos-SD1-for 5'-AGCGCAAATTGCAGAATTACAAAGA-3', PB-cos-SD1-rev 5'-GGTTAGAGAGGATAAGCCGCATGTT-3', DB-cos-SD1-for 5'-GATCCCGCACATTTGTTCATTAGAG-3', DB-cos-SD1-rev 5'-CAAAAGCTTGAATTCTGTGCCTCAGT-3'
Detection of derived CAGGG repeat sequences in the Y-chromosomal cosmid LLOYNC03 "M" 22E01
We used RepeatMasker http://www.repeatmasker.org/ and Tandem Repeats Finder  to search for CAGGG repeat sequences within the Y-chromosomal CHEK2 duplicon containing cosmid LLOYNC03"M" 22E01. Both analyses failed to identify CAGGG repeat sequences in the derivative CHEK2 duplicon and in both adjacent derivative duplicons, which are the IGL@- (Immunoglobulin lambda@ locus-) and the NHEDC1- (Na+/H+ exchanger domain containing 1-) duplicons. Nevertheless, RepeatMasker and Tandem Repeats Finder analysis revealed the presence of extensive CAGGG repeat sequences within the ancestral IGL@ locus in 22q11.21 (NT_011520: 1994740–2060046). Subsequent pairwise alignment of both ancestral and the derivative Y-chromosomal IGL@ loci disclosed a more diverged CAGGG repeat like sequence. By using the DnaSP Ver.4.10.9 software 72% nucleotide sequence identity was detected between the CAGGG repeats present within the ancestral IGL@-duplicon (NT_011520: 2033670–2035327) and the derivative CAGGG repeats in Yq11.1/Yq11.21 (NT_113819: 398233–403886).
Blood samples and cell lines
The following blood samples from non-related individuals were used for qPCR analysis: blood samples from the chimpanzees (Pan troglodytes, PTR) Max (LN: 444) and Fritz (LN: 776, wild-born) were obtained from the Zoologisch-Botanischer Garten Wilhelma Stuttgart (Germany). The blood sample from chimpanzee Marcel (LN: 39) was obtained from TNO Primate Centre Rijswijk (Netherlands). Lowland gorilla (Gorilla gorilla gorilla, GGO) blood samples of Jangu (LN: 315), Gaidi (LN: 916, wild-born) and Fritz (LN: 673, wild-born) were obtained from the Zoo Wuppertal, the Zoo Leipzig and the Tiergarten Nürnberg, respectively. Two blood samples of the Bornean orangutans (Pongo pygmaeus pygmaeus, PPY) Thai (LN: 866) and Napoleon (LN: 1005) were obtained from the Zoo Duisburg (Germany) and the Zoo Studen (Switzerland), respectively. Blood samples from two rhesus macaque individuals (Macaca mulatta, MMU; LN: 1053 and 1054) and one hamadryas baboon individual (Papio hamadryas, PHA; LN: 496) were obtained from the Deutsches Primaten Zentrum Göttingen (Germany).
Skin tissue of a pig-tailed macaque (Macaca nemestrina, MNE) was provided by the Deutsches Primaten Zentrum Göttingen (Germany) and was used to establish a fibroblast cell line. For each species one of the above listed blood samples and the fibroblast cell line were used for FISH analysis. Lymphoblastoid cell lines of the white-cheeked crested gibbon (Nomascus leucogenys, NLE) and the common marmoset (Callithrix jacchus, CJA) were kindly provided by S. Müller (Munich) and were used for FISH analysis.
Fluorescence in situ hybridization (FISH)
FISH analysis of metaphase spreads derived from lymphocytes or lymphoblastoid and fibroblast cell lines from non-related human (Homo sapiens, HSA) and non-human primate males was performed. Prior to FISH, the slides were treated with RNase followed by pepsin digestion as described . FISH was carried out following the protocol described previously . Chromosome in situ suppression was applied to clones from the human fosmid library WI-2 (WI2-1621D20, WI2-819H21) and from the Y chromosome specific cosmid library LLOYNCO3"M" (LLOYNCO3"M"22E01). Human whole-chromosome painting (WCP) libraries  were used to unequivocally assign hybridizing signals to orthologous regions in lesser apes, Old World monkeys and New World monkeys. pMR100, a mouse-derived rDNA-containing plasmid, was used to tag the Old World monkey marker chromosome. After FISH the slides were counterstained with DAPI (4',6-diamidino-2-phenylindole; 0.14 μg/ml) and mounted in Vectashield (Vector Laboratories). Preparations were evaluated using a Zeiss Axiophot epifluorescence microscope equipped with single-bandpass filters for excitation of red, green, and blue (Chroma Technologies, Brattleboro, VT). During exposures, only excitation filters were changed allowing for pixel-shift-free image recording. Images of high magnification and resolution were obtained using a black-and-white CCD camera (Photometrics Kodak KAF 1400; Kodak, Tucson, AZ) connected to the Axiophot. Camera control and digital image acquisition involved the use of an Apple Macintosh Quadra 950 computer.
FASTA formatted sequence files used to generate phylogenetic trees were extracted from the corresponding GenBank accession numbers. Sequence alignments were built by using CLUSTALW (version 1.82) , and neighbor-joining phylograms created by using MEGA (Molecular Evolutionary Genetic Analysis) v4.0 http://www.megasoftware.net. Neighbor-joining analysis was used with complete deletion parameters and bootstrap (1,000 iterations) to provide confidence of each branching point in the phylogenetic trees. Neighbor-joining methods were chosen as they are amenable to calculating divergence times between sequence taxa. We estimated the number of substitutions per site per year by correcting the divergence times for multiple substitutions using Kimura's two-parameter model . As the rates of nucleotide substitution vary for pseudogenic sequences, the rate of nucleotide substitution was calibrated based on orthologous sequence comparisons using a divergence of 25 Mya for macaque-human divergence . Duplication timing events were calculated by applying the equation r = k/2 T , where r is the rate of nucleotide changes per bp per yr, k is the distance calculated between the ancestral and paralogous sequences, and T is the time of divergence of the molecules.
Interspecies quantitative PCR was carried out using primers specific for CHEK2 exon 14. Primers were designed with the assistance of the Promega Plexor Primer Design Software. The following primer sequences were used: CHEK2-exon14-F 5'-GGACCTTGTCAAGAAGTTGTTGGT-3', CHEK2-exon14-R 5'-GGTGTCTTAAGGCTTCTTCTGTCGTA-3', CFTR-F 5'-CGCGATTTATCTAGGCATAGGC-3' and CFTR-R 5'-TGTGATGAAGGCCAAAAATGG-3'
We used the ABI Prism 7900 HT system (Applied Biosystems) for real time detection. Reactions contained 0.25 μM of each primer and 5 μl of QuantiTec SYBR® Green PCR Master Mix (Quiagen) in a total of 10 μl. Assays included DNA standards at a final concentration of 5.0, 2.5, 1.25, 0.625 and 0.3125 ng/μl, a no-template control, or 1 ng/μl of the species DNA in two replicates. Cycling conditions were 50°C for two minutes, 95°C for 15 minutes, and 40 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec.
To avoid the generation of non-specific products, a melting curve analysis of products was routinely undertaken following the amplification. A standard curve was constructed by plotting the cycle number (Ct), at which the amount of target in standard dilutions reaches a fixed threshold, against the log of the amount of starting target. For standard curve construction genomic DNA from the rhesus macaque MMU#13577 was used as a CHEK2 single copy reference. The CHEK2 single copy status in the rhesus macaque genome was verified by both FISH and in silico analysis. Absolute quantification of copy number in the different species was subsequently done by interpolation of the threshold cycle number (Ct) against the corresponding standard curve. Copy numbers of the test genes in primate samples were normalised to the copy number of the CFTR gene (cystic fibrosis transmembrane conductance regulator), which serves as a control representative of a single gene per haploid genome . CFTR primers perfectly match the CFTR gene in all targeted species genomes. The ratio of the CHEK2 copy number to CFTR copy number in each sample normalised the results with respect to differing starting quantity and quality of the template DNA in each reaction . Standard errors of the normalised CHEK2 copy numbers were calculated from the standard deviations of the values of the CFTR and CHEK2 genes using the formula provided by the user menu (ABI Prism 7700 Sequence Detection System, User Bulletin no.2 1997, p.34). Comparisons between the mean values were performed using the Student unpaired t-test. A P-value <0.001 was considered significant.
Identification and comparative FISH of a CHEK2duplicon containing Y-chromosomal cosmid probe
Comparative FISH results of cosmid clone LLNLYC03"M" 22E01 from Yq11.1/q11.21
Comparative FISH with CHEK2fosmid probes from 22q12.1 in anthropoids
Comparative FISH results of fosmid clone WI2-1621D20 from 22q12.1
To verify the single-copy status of the non-duplicated portion of the CHEK2 locus, we performed FISH with the fosmid probe WI2-819H21 (G248P81285D11; Figure 2A). In all analysed primate species, including human, this probe hybridized to a single genomic localisation corresponding to the human 22q12.1 orthologous regions. In conclusion, our comparative FISH results with fosmid probe WI2-1621D20 (Figure 2B, Table 2) show the single-copy status of CHEK2 in all tested New World and Old World monkeys and in the white-cheeked crested gibbon. While a first CHEK2 duplication event was detected in the orangutan, a burst of duplication, giving rise to the complex signal pattern, occurred before the radiation of African great apes. Except for chromosomes 16 and Y all signals present in non-human primates were also detected in the orthologous human regions.
Comparative in silico analysis of CHEK2duplicons in anthropoids
To elucidate the molecular evolution of the derivative CHEK2 duplicons we investigated their flanking paralogous sequences. All known CHEK2 duplicons extend into the same proximal duplicated sequence, indicating a single initial pericentromeric "seeding" event. Subsequent pericentromeric "swaps" of the larger duplicon cassette led to the CHEK2 duplicon distribution observed in the human genome. The ancestral location of the duplicon proximal to all CHEK2 duplicons resides within the IGL@ locus in 22q11.21. The duplicated sequences extending beyond the distal CHEK2 duplicon junctions are not identical for all derivative duplicons. While CHEK2 duplicons 16p11.2a and 15q11.2 show the same distal homology, duplicons 2q11.1/2q11.2, 10p11.1, 16p11.2b, 22q11.1, Yq11.1/Yq11.21 and Chr_random share a different distal duplicated sequence. Thus, the latter set of duplicons may have arisen by a duplication of the 16p11.2b duplicon, followed by a LINE1 element integration and successive pericentromeric exchange.
Subsequently, we used the same in silico approach to determine the CHEK2 duplicon architecture of the chimpanzee genome (build2). Chromosomally assigned duplicons in the chimpanzee genome showed chromosomal designations corresponding to the human genome locations. Duplicons were detected on chimpanzee chromosomes 10, 15, 16, 22 and Y (Figure 3B), but not on chromosome 2A. Two duplications containing almost the entire CHEK2 duplicon and 11 smaller duplicon fragments were not chromosomally assigned in the current chimpanzee whole genome assembly. Similar to the human genome the largest CHEK2 duplicon is assigned to chromosome 16 and a slightly shorter CHEK2 duplicon is located on chromosome 15. In contrast to the human genome the chromosome 15 duplicon contains no deletion, indicating that this deletion is human specific. In addition, duplicons on chimpanzee chromosomes 10 and 15 contained small internal duplications and chromosome 16 duplicons seemed to be more fragmented (Figure 3B). These findings might be explained by chimpanzee-specific rearrangements or, more likely, by the inherent problem of generating highly reliable contiguous sequence assemblies in regions enriched in SDs.
Comparative in silico analysis (BLAT search http://genome.ucsc.edu/ of the orangutan (ponAbe2) and rhesus macaque (rheMac2) whole genome assemblies using the human duplicated CHEK2 sequence yield just one copy in the respective genomes on their orthologous chromosome 22. No CHEK2 duplicon was found on orangutan chromosome 16 by this approach, but this may be due to the underrepresentation of segmentally duplicated sequences within pericentromeric regions in the whole genome assemblies . The unduplicated status of CHEK2 in the rhesus macaque genome assembly is concordant with our FISH results obtained with fosmid WI2-1621D20.
Phylogenetic reconstruction of CHEK2duplicon events in anthropoids
Detection of inter-species copy number variations of CHEK2by quantitative PCR
CHEK2, which is essential for genomic stability , is known to be a multiorgan cancer susceptibility gene and is frequently analysed in tumour diagnostics, e.g. of breast, colorectal and prostate cancer [61, 62]. CHEK2 is one of a multitude of genes known to be part of SDs [10, 63]. Our detailed three-pronged approach clearly demonstrates that CHEK2 duplicons show a high degree of both copy number variation and sequence identity. Furthermore, there is strong evidence, that not all duplicons in the human genome have been sequenced yet. Thus, molecular diagnostics of CHEK2 inside the duplicated region might be hampered by the individual-specific set of CHEK2 duplicons. One previously published variant (1422delT) of the functional CHEK2 gene, was thought to predispose to Li-Fraumeni syndrome , but subsequently was shown to be the genomic sequence of a CHEK2 duplicon . To avoid such diagnostic pitfalls in the analysis of duplicated disease related genes, it is essential to close the still existing gaps in the human genome sequence by closely examining segmentally duplicated regions. Additionally, copy number and sequence variation within SDs might require further efforts to adapt the diagnostic settings to different ethnic backgrounds.
Furthermore, our detailed CHEK2 analysis revealed its highly dynamic nature during anthropoid evolution. Both, FISH and phylogenetic analyses suggest the first duplication event to have occurred before the radiation of the great ape species. Extensive pericentromeric exchange and intrachromosomal duplication events led to a burst of CHEK2 duplications before the radiation of the African great apes followed by lineage specific rearrangements creating species-specific distribution patterns in great apes and human. In conclusion, our analysis of the CHEK2 duplicon evolution reveals, that SDs contribute to inter-species variation.
We thank Christine Hodler for technical assistance, and Alexander Craig for stylistic revisions of the manuscript. Supported by grants from the Deutsche Forschungsgemeinschaft (Sche 214/8-1).
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