MBE Advance Access originally published online on September 12, 2008
Molecular Biology and Evolution 2008 25(12):2601-2613; doi:10.1093/molbev/msn202
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Research Articles |
How Segmental Duplications Shape Our Genome: Recent Evolution of ABCC6 and PKD1 Mendelian Disease Genes
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary
E-mail: aranyi{at}enzim.hu.
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Abstract |
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The completion of the Human Genome Project has brought the understanding that our genome contains an unexpectedly large proportion of segmental duplications. This poses the challenge of elucidating the consequences of recent duplications on physiology. We have conducted an in-depth study of a subset of segmental duplications on chromosome 16. We focused on PKD1 and ABCC6 duplications because mutations affecting these genes are responsible for the Mendelian disorders autosomal dominant polycystic kidney disease and pseudoxanthoma elasticum, respectively. We establish that duplications of PKD1 and ABCC6 are associated to low-copy repeat 16a and show that such duplications have occurred several times independently in different primate species. We demonstrate that partial duplication of PKD1 and ABCC6 has numerous consequences: the pseudogenes give rise to new transcripts and mediate gene conversion, which not only results in disease-causing mutations but also serves as a reservoir for sequence variation. The duplicated segments are also involved in submicroscopic and microscopic genomic rearrangements, contributing to structural variation in human and chromosomal break points in the gibbon. In conclusion, our data shed light on the recent and ongoing evolution of chromosome 16 mediated by segmental duplication and deepen our understanding of the history of two Mendelian disorder genes.
Key Words: primate evolution • nonallelic homologous recombination • morpheus • segmental duplication • copy number variation • Mendelian disease genes
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Segmental duplications younger than 35 Myr are a major constituent of the human genome, comprising about 5% of our genomic sequence (Bailey and Eichler 2006
). They contribute to genomic instability, development of disease, and the evolution of new genes (Mazzarella and Schlessinger 1998; Emanuel and Shaikh 2001; Bailey and Eichler 2006). Recent analysis of human segmental duplications has shown that they are frequently organized in clusters, which are composed of a complex mosaic of duplication modules. These modules are blocks of duplicated sequence present in various numbers and sometimes in different order relative to each other. Based on copy number and sequence divergence, they can generally be categorized as "core" and "surrounding" duplicons (Johnson et al. 2006; Zody, Garber, Adams, et al. 2006; Zody, Garber, Sharpe, et al. 2006; Jiang et al. 2007). Core modules show the highest copy number and sequence divergence, and it has been put forward that they are genomically unstable regions prone to duplication (Jiang et al. 2007). Furthermore, in some cases, core duplicons code for primate-specific genes. Accordingly, the increased number of these genes due to the duplications could have conferred a selective advantage favoring their recent rapid expansion (Jiang et al. 2007). This hypothesis also suggests that the instability and proliferation of the core duplicon drive the duplication of the surrounding genomic regions, essentially as a secondary effect. However, it has not been analyzed if the accompanying duplication of the flanking regions has any functional consequences and, if so, how these are manifested. In the present study, we answer these questions by an in-depth study of ABCC6 and PKD1 as model genes. Both genes are located on the short arm of chromosome 16, and mutations in these genes are responsible for Mendelian disorders. Their genetic analysis is complicated by the presence of multiple nonprocessed, truncated copies of both ABCC6 and PKD1 along chromosome 16p.
All ABCC6 and PKD1 pseudogenes (hereafter labeled as ABCC6-
or PKD1-) are positioned within large duplication clusters on chromosome 16p. These duplication clusters have been extensively studied, and it has been etermined that they are mosaics of smaller duplication modules (named low-copy repeat [LCR] 16a–x) (Loftus et al. 1999; Johnson et al. 2006). Among these modules, LCR16a has been suggested to act as core duplicon (Johnson et al. 2006). In agreement with the core region model, LCR16a is the most common duplicon on chromosome 16 (Loftus et al. 1999; Johnson et al. 2006) and encodes a gene known as morpheus that has been under strong positive selection in primates (Johnson et al. 2001).
ABCC6 codes for the ATP-binding cassette transporter protein ABCC6/MRP6. The gene is located at 16p13.11, and its mutations are responsible for pseudoxanthoma elasticum (MIM 264800
[OMIM]
) (Le Saux et al. 2000; Ringpfeil et al. 2000; Bergen et al. 2007), a recessive disorder characterized by elastic tissue fragmentation and calcification. To date, 
180 pseudoxanthoma elasticum-associated mutations have been published (Plomp et al. 2008). These mutations were identified mainly in the coding region and although they can be found throughout the gene, it has been observed that they cluster in exons corresponding to the C-terminal part of the protein. The functional gene has two pseudogenes (Cai et al. 2001; Germain 2001; Pulkkinen et al. 2001) that are expressed at low levels (Cai et al. 2001; Aranyi et al. 2005; Piehler et al. 2008) and are positioned centromeric (ABCC6-1) and telomeric (ABCC6-2) to ABCC6. Both pseudogenes share a high degree of similarity (99%) with the functional gene but are truncated in the fourth and ninth intron, respectively.
PKD1 encodes polycystin-1, a membrane-spanning protein, which is required for the cell calcium response of ciliated cells (Nauli et al. 2003; Xu et al. 2003). PKD1 is positioned at 16p13.3, and mutations in the gene are responsible for 85% of autosomal dominant polycystic kidney disease (MIM 173900
[OMIM]
), the most common hereditary renal disorder (with a prevalence of 1:1,000) (Consortium 1994). A total of 270 mutations have been reported for PKD1, the majority (67%) being nonsense, frameshift, or splicing events and 33% missense or other in-frame events. However, no correlation between mutation type and disease has been found, although mutations toward the 5' end of the gene seem to be more detrimental (Rossetti and Harris 2007). The first 33 exons of PKD1 are reiterated several times along chromosome 16 (Consortium 1994; Loftus et al. 1999; Bogdanova et al. 2001), and this has made the analysis of this region extremely difficult. The precise number of PKD1 pseudogenes (also known as homologous genes) is unknown, but they are transcribed (Consortium 1994; Loftus et al. 1999; Bogdanova et al. 2001), and a major effort has been made to develop mutation detection strategies that are specific for the disease-causing gene (Peral et al. 1997; Roelfsema et al. 1997; Thomas et al. 1999; Phakdeekitcharoen et al. 2001; Rossetti et al. 2001).
Currently, although both ABCC6 and PKD1 are clinically important genes, little effort has been made to understand the effect of their partial duplication, with the exception of anecdotal reports of gene conversion (Watnick et al. 1997, 1998; Cai et al. 2001). Here, we annotate ABCC6 and PKD1 pseudogenes to the duplication modules LCR16x and LCR16b, respectively, which were previously suggested to have arisen through LCR16a-dependent duplication. As a consequence, the pseudogenes of PKD1 and ABCC6 have contributed to the birth of new genes and the duplicated segments are involved in recombination-based gene conversion and rearrangements in humans, whereas in the white-cheeked gibbon (Nomascus leucogenys), similar rearrangements can potentially lead to macroscopic chromosomal break points.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Identification and Mapping of Pseudogenes, Bacterial Artificial Chromosome/Fosmid/Cosmid Clones, and Expressed Sequence Tags
To identify and map pseudogenes in LCR16a-associated duplication clusters, we searched for ABCC6 (NM_001171 [GenBank] ), PKD1 (NM_000296 [GenBank] ), and morpheus (NM_006985 [GenBank] ) sequences by applying systematic BLAT searches (Kent 2002
) to the March 2006 (hg18) genome assembly at the University of California Santa Cruz Human Genome Browser (Kent et al. 2002). We verified our results by comparing them to entries in the Human Genome Segmental Duplication Database (Cheung et al. 2003) and the Segmental Duplication Track of the UCSC Genome Browser (Bailey et al. 2001). To find DNA clones from human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), gibbons (Hylobatidae), or the white-cheeked gibbon (N. leucogenys), we performed nucleotide Blast searches at the National Center for Biotechnology Information Web server using sequences as indicated in the text. We selected the nr (for finished clones) and the htgs (for unfinished high-throughput genomic sequences) databases when searching for bacterial artificial chromosome (BAC), fosmid, and cosmid clones or the expressed sequence tags (ESTs) database to identify transcripts. We did not use the current chimpanzee genome assembly because preliminary analysis showed that the region of interest is still incompletely assembled, containing several gaps and partial ABCC6 and PKD1 genes are assigned to random chromosome fragments.
To map identified clones to the reference human genome, we used BLAT searches as described above. To unambiguously map duplicated sequence on the chromosome, we either relied on unique sequences flanking the duplications or, if this was not applicable, the sequence had to be consistently similar to a single continuous chromosomal segment. To avoid misinterpretation of assembly artifacts or unique clones, we only accepted BAC, fosmid, or cosmid clones (in the case of human and gibbon chromosomal rearrangements) or EST sequences as correct if there was a "redundancy of information": that is, either we found several overlapping but nonidentical clones from a single library or the same rearrangement/transcript was identified in several different libraries. In the case of structural rearrangements, we also accepted if our data from a single library were in agreement with previous reports of structural rearrangements or copy number variation.
Phylogenetic Reconstruction
For phylogenetic reconstruction of human and chimpanzee ABCC6 and PKD1-like genes, we aligned the largest common fragment from the genes and pseudogenes with ClustalW (Chenna et al. 2003), using sequence from the human reference genome and chimpanzee BAC clones. We then selected the best model of evolution for these alignments according to the Akaike and Bayesian information criteria scores of various models, as implemented by TOPALi v2 (Milne et al. 2004). We used a K81 model with unequal nucleotide frequencies, invariant sites, and gamma distribution to establish the sequence distances and to construct unrooted maximum likelihood phylogenetic trees using TOPALi v2. Trees were tested by bootstrapping (1,000 repeats).
To determine sequence divergence, we used separate multiple alignments of exons and introns to calculate pairwise distances. We established statistical significance using Student's t-test.
Identifying Gene Conversion Events
We identified nucleotide differences between pseudogenes and genes by first aligning the corresponding genes and pseudogenes using reference human genome sequence. Next, we collected sequence variants in the duplicated region of PKD1 and ABCC6 from all mutational reports that covered this region and had used a method which distinguished between the genes and their pseudogenes (e.g., using gene-specific primers or long-range polymerase chain reaction [PCR]-utilizing primers anchored in single-copy regions of the genes). The PKD1 variants were verified by comparison with the PKD1 mutation database (Gout et al. 2007), which contained complementary or corrected data for three mutations (c.6598C>T substituted p.R2200C, c.8344G>A substituted c.8345G>A, and c.10064C>T substituted p.P3355L). All sequence variants of the disease-causing genes that fell within the duplicated region were then compared with our alignment to see if it matched a difference between the gene and one of its pseudogenes. If so, we considered it a potential gene conversion event. To establish definite examples of gene conversion, we further filtered the list of potential events for two or more co-occurring variants that were in perfect agreement with the sequence of one or more of the pseudogenes. All variants are described according to the current nomenclature standards (den Dunnen and Antonarakis 2001).
Identifying Repeat Sequences and Open Reading Frames within the Pseudogene Transcripts
To find repetitive elements in the transcripts, we screened them using CENSOR (Kohany et al. 2006). The internal duplication within the LCR16a element adjacent to PKD1-2 was discovered by performing self–self comparisons of the LCR16a sequences using the dot-plot program dotter (Sonnhammer and Durbin 1995). Transcripts were in silico translated in all three forward frames using Transeq at the European Bioinformatics Institute homepage.
Experimental Validation of the Presence of the ABCC6-1 Transcript
Cell culture of HepG2 cells, RNA extraction, and reverse transcription was carried out as described previously (Aranyi et al. 2005). Human liver cDNA was obtained from the RACE-ready liver cDNA kit (Ambion, Austin, TX). The transcript was amplified using nested PCR. Primers were designed and in silico tested using the BiSearch Web server (Aranyi et al. 2006). In HepG2 cells, we used primers F1 (5'-GGC TCA AGG TGC TGT AGA TGA GG-3') combined with R1 (5'-GGC TTG GCG TTC TTG AGG TTA-3') for the first round and F2 (5'-GGT GTA CAG AAA GGC ATC CAC AG-3') combined with R2 (5'-TTC TTG AGG TTA ATG CTG CGT G-3') for the second round of PCR. For liver cDNA, we used F1 as outer and F2 as inner forward primers in combination with the reverse primers provided in the kit. PCR conditions were as follows: 1.5 mM final MgCl2 concentration, 92 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s for 35 cycles. PCR products from liver cDNA were subcloned by T/A cloning into the pGEM-Teasy vector (Promega, Mannheim, Germany) and sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and analyzed using ABI PRISM 310 sequencer.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Selection and Validation of PKD1 and ABCC6 as Model Genes
To analyze the effect of duplication mediated by core regions, we selected LCR16a-associated segmental duplications as model system because the function of LCR16a as core had been extensively studied previously (Loftus et al. 1999
; Johnson et al. 2006; Jiang et al. 2007). An overview of the literature and existing databases revealed the presence of multiple PKD1- and ABCC6-like sequences in LCR16a-associated duplications. PKD1 and ABCC6 are also the genes with the highest clinical relevance in the region and have the best-characterized duplications, probably due to their functional importance.
First, we mapped the genes and their pseudogenes (fig. 1A) by systematically searching for PKD1 and ABCC6 sequences in the reference human genome (see Materials and Methods). Our results regarding ABCC6 confirmed data from the literature, that is, we found the full-length gene and the two pseudogenes (supplementary table 1, Supplementary Material online) sharing 98.9% (98.7–99.2%) sequence similarity. We found that all three sequences are within LCR16x duplicons, near to LCR16a elements (fig. 1B). The LCR16x module has been reported to duplicate in an LCR16a-dependent manner (Johnson et al. 2006). By determining this association of ABCC6 with LCR16a, we are able to link it with a previously proposed mode of duplication on chromosome 16 and thus, for the first time, provide a potential explanation for the duplication of this gene.
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The PKD1-like genes had never been mapped before, and their number had only been ambiguously determined (Consortium 1994
; Loftus et al. 1999; Bogdanova et al. 2001). We established that the reference human genome contains six truncated PKD1 pseudogenes (supplementary table 1 [Supplementary Material online] and fig. 1A). This is consistent with data from Bogdanova et al. (2001), who also reported the existence of six or more PKD1 homologous genes. However, except for PKD1-2 and PKD1-4, it was not possible to unambiguously determine a clear one-to-one relationship between the previously reported pseudogenes and those present in the consensus human genome (supplementary table 1A, Supplementary Material online). On average, the PKD1 pseudogenes share 97.7% (97.6–97.8%) similarity with the functional gene and 98.8% (98.3–99.8%) identity with each other. We annotated them to the LCR16b elements at 16p13.11 and 16p12.3 (fig. 1B). LCR16b module has also been reported to duplicate in an LCR16a-dependent manner (Johnson et al. 2006). Surprisingly, in the case of PKD1, we found that the pseudogenes are positioned in proximity to LCR16a elements, but the gene itself is not (fig. 1B). We therefore presumed that the initial duplication of PKD1 must have either occurred independently of LCR16a or the LCR16a element associated with the PKD1 gene was later deleted. To resolve this question, we examined the repertoire of LCR16a elements in the great apes and located LCR16a sequence close to the 5' end of PKD1 in gorilla (BAC clone AC144881
[GenBank]
). This supports the notion that the initial duplication of the gene was mediated by an LCR16a element, which was subsequently deleted. Furthermore, analysis of the existing chimpanzee BAC clones revealed the presence of at least three LCR16a-related ABCC6 pseudogenes and two PKD1 pseudogenes in the chimpanzee genome (supplementary table 1B, Supplementary Material online). In an attempt to reconstruct the relationship of the human and chimpanzee pseudogenes, we compared their size and structure and assembled phylogenetic trees where possible (fig. 1C). Our data showed that overall the chimpanzee pseudogenes are smaller, have other break points than those observed in human, and they are positioned within more complex duplication clusters than in human. On the phylogenetic trees, the human and chimpanzee sequences form separate branches (fig. 1C). Collectively, structure and divergence suggest that the ABCC6 and PKD1 pseudogenes in human and chimpanzee were created independently, which further testifies to the high mobility of the LCR16a-associated genomic sequences. Accordingly, we devised a possible evolutionary scenario based on the core region hypothesis (see supplementary file 1, Supplementary Material online) to explain the partial duplication of PKD1 and ABCC6 during primate evolution.
Altogether, these data validate PKD1 and ABCC6 as model genes for the study of functional relevance of LCR16a-mediated segmental duplications: they are located within LCR16a duplication clusters, duplication seems to have been mediated by LCR16a as core, and because they are Mendelian disorder genes, a large data set of sequence variants is available.
Consequences of LCR16a-Mediated Segmental Duplications
PKD1 and ABCC6 Pseudogenes Encode Novel Chimeric Transcripts
When analyzing the consequences of LCR16a-mediated segmental duplications, we first hypothesized that partial duplications of ABCC6 and PKD1 might have resulted in the creation of new genes because it is known that juxtapositioning of partial coding regions (due to duplication or chromosomal rearrangement) can lead to the formation of novel fusion genes resulting in evolutionary innovation (Bailey et al. 2002; Bridgland et al. 2003; Paulding et al. 2003) or pathological consequences (Gasparini et al. 2007; Krivtsov and Armstrong 2007).
Because it is known that the pseudogenes are expressed, we searched the EST database with fragments of PKD1 and ABCC6 sequence. We identified clones from several PKD1 pseudogenes and ABCC6-1, present in a range of tissues (fig. 2; supplementary table 2, Supplementary Material online). Furthermore, we observed that the majority of these clones are chimeric transcripts: only one part is encoded by the respective pseudogene, whereas the other half is derived from duplications adjacent to LCR16b/LCR16x.
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A more detailed inspection of the PKD1 pseudogene transcripts showed that they are in fact fusions of the pseudogenes and copies of the morpheus gene, which is encoded by LCR16a (fig. 2A). In most cases, we could not define from which of the PKD1 pseudogenes the EST was transcribed due to the high degree of similarity between the pseudogenes and the low sequence quality of some of the ESTs. Exceptions to this observation are the various transcripts derived from PKD1-
2 because an internal duplication within the LCR16a region results in a duplication of the first morpheus exon. However, none of the PKD1-2 transcripts were supported by more than one EST clone; therefore, we did not consider them reliable according to our standards. For the remaining transcripts, we found that several splice variants exist, and some of these include repeat elements (fig. 2). In addition, we found that most of the PKD1 pseudogene transcripts retained PKD1 intron 31 and thus exon 31, intron 31, and exon 32 form a single exon in these mRNAs. This retained intron correlates with the presence of a 19-bp deletion within intron 31 of the corresponding pseudogenes compared with PKD1. To better understand the potential function of these transcripts, we tested whether they contain any open reading frames. We found that in all cases, the longest open reading frames were derived from the morpheus part of the transcript, whereas the PKD1-derived part contained multiple stop codons in all frames. We therefore concluded that the PKD1 pseudogene transcripts do not fulfill a protein-coding function. As an alternative method to establish potential functionality, we compared the degree of sequence divergence in exonic and intronic regions. We assumed that if the exons had no function, then their rate of divergence would not differ from that of the introns. We therefore compared the pairwise distances of the exonic and intronic regions and found that compared with the functional PKD1 gene the pseudogene exons had diverged at a significantly faster rate than introns (average pairwise distance 0,0024 and 0,0019, respectively, P = 0.002). A similar tendency could be seen when we compared pseudogenes with each other, but the difference was not statistically significant. Conclusively, this suggests that PKD1 pseudogene transcripts have no protein-coding function, but they may have regulatory function as suggested by the fact that the exonic regions have evolved at a different rate than the introns.
In the case of the ABCC6 pseudogenes, we identified five clones from various tissues showing that the ABCC6-1 transcript is complemented by a further exon from a recent duplication adjacent to the pseudogene (fig. 2B). Unlike LCR16a, the duplication adjacent to ABCC6-1 does not have any coding potential by itself, as observed by the lack of cDNAs and RefSeq genes from this region. Because there were only a small number of supporting EST clones, we validated the presence of the ABCC6-1 transcript experimentally. Using specific primers, we amplified and sequenced the transcript in human liver and HepG2 cDNA, showing that it is indeed expressed in liver tissue (fig. 2B).
In agreement with Pulkkinen et al. (2001), we found that the functional ABCC6 translational frame is disrupted by a single base insertion in the second exon. This causes a frameshift and subsequently multiple stop codons. However, if translation initiated at the third ATG of the pseudogene transcript, an open reading frame coding for 345 amino acids could be obtained. In this event, the stop codon is positioned in the ninth exon of the transcript, and the exon derived from the adjacent duplicon does not form part of the open reading frame.
These data therefore confirm our first hypothesis that pseudogenes of both PKD1 and ABCC6 can potentially participate in the evolution of new genes, although it will need further investigation to clarify their potential physiological function.
PKD1 and ABCC6 Pseudogenes Can Cause Gene Conversion
Next, we postulated that the presence of multiple copies of nearly identical gene sequences could lead to nonallelic homologous recombination and thus pseudogene-mediated gene conversion. Indeed, gene conversion seemed plausible because it is believed that the sequence identity required for this process is usually >95% (Chen et al. 2007), which conforms well to the sequence similarity we had found between ABCC6, PKD1, and their respective pseudogenes (see above). To test this idea, we performed a thorough review of the mutational literature and mutation databases and searched for sequence alterations of the functional PKD1 and ABCC6 genes that are identical to the pseudogene sequences. We found 54 polymorphisms and mutations in PKD1 and 13 in ABCC6 that might potentially be the result of gene conversion (supplementary tables 3 and 4, Supplementary Material online). The simplest way to explain the higher number of potential PKD1 gene conversion cases is the larger number of PKD1 pseudogenes and a greater divergence of PKD1 from its pseudogenes, which are also longer than the ABCC6 pseudogenes. Detailed characterization of the potential gene conversion events shows that although the majority affect the exonic region of the genes (84% and 81% for ABCC6 and PKD1, respectively), only a small portion was found to have disease-causing potential (15% and 12%). The ABCC6 and PKD1 exonic sequence variants can be grouped as samesense (36.4%, 41%), missense (54.5%, 50%), nonsense (9.1%, 6.8%), and deletion (0%, 2.2%). Also 30.8% and 27.8% of the potential gene conversions can be explained by C > T transitions at CpG positions, which are known to be mutational hot spots due to the spontaneous deamination of 5mC nucleotides (Pfeifer et al. 2000). To rule out the possibility of mistakenly annotating sites with higher-than-average mutation rates as gene conversion, we finally only accepted clusters of polymorphisms/mutations with a pattern that perfectly matched one or more of the pseudogenes as definite examples (see Materials and Methods). However, potentially there are far more real gene conversion events among the list of putative variants because we probably excluded a large number of positives using these stringent criteria. This assumption is based on the observation that the average size of gene conversion tracts in mammals is relatively small (200 bp–1 kb in length [Chen et al. 2007]). Due to the high degree of sequence similarity, it is possible that only a single mismatch would be included in a region of such size, which would therefore not be called in our list of definite events.
As shown in table 1, in total, there were six definite gene conversion events for PKD1 and two for ABCC6, which were all within the expected size range. With one exception, these were clusters of mismatches, the exception being a 19-bp deletion in intron 31 of PKD1, reportedly causing retention of intron 31 (Peral et al. 1997; Rossetti et al. 2001), just as we had observed for the expressed pseudogenes. We also observed that six out of eight of these definite gene conversion events contained at least one disease-causing mutation within the mismatch cluster. On the whole, these data strongly suggest that the ABCC6 and PKD1 duplication can be involved in nonallelic homologous recombination and therefore cause gene conversion.
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LCR16-Associated Duplications Are Involved in Genomic Rearrangements in Human
Nonallelic homologous recombination is also thought to cause more substantial genomic rearrangements, such as deletions, insertions, and inversions (Shaw and Lupski 2004
). Therefore, based on our second hypothesis on the increased probability of nonallelic homologous recombination between the segmental duplications, we tested whether the regions containing PKD1- and ABCC6-like sequences were involved in structural variations as well. To do this, we first searched for BAC, fosmid, and cosmid clones containing sequences from these regions. We then mapped the clone sequences back to the reference human genome and looked for discrepancies that might represent genomic rearrangements. Using this approach, we were able to unambiguously map at least four different rearrangements affecting this region (fig. 3). Three of the four rearrangements were present in at least two of the following libraries: the RPCI-11 BAC (Osoegawa et al. 2001), the Cal Tech D BAC, the WI2 fosmid, or the Los Alamos chromosome 16 cosmid (LA16c) library. This shows that the same rearrangement is sometimes present in multiple individuals. Because the RPCI-11 BAC library was used to construct the bulk of the chromosome 16 reference sequence (Martin et al. 2004) and it originates from a single individual, it seems probable that this person was heterozygous for some of the rearrangements.
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Rearrangement A was found in BAC clones from the RPCI-11 and the CTD BAC library. The BAC clones carry a 0.4-Mb deletion relative to the consensus genome sequence, and one of the segments bordering on the deletion is inverted. The break point of the rearrangement is positioned within a
70-kb duplicon, which is present in inverse orientation in the two segments that harbor the rearrangement. LCR16a forms part of this 70-kb duplicon. Rearrangements B and C were identified in clones from the RPCI-11, the WI2, and the LA16c libraries. Both rearrangements consist of a deletion and an inversion of the neighboring sequence, although the deletions are smaller (30 and 50 kb, respectively) than in rearrangement A, and there is no overlapping duplicated sequence spanning the break points. However, in rearrangement B, the break point is immediately adjacent to an LCR16a element. Both rearrangements B and C were identified previously in a study using fosmid DNA paired-end mapping (Tuzun et al. 2005). Rearrangement D shows the most complex rearrangement pattern and is most probably the result of multiple events. It is present in a single library (LA16c) but supported by several different clones. In this case, both rearrangements B and C occur in a single BAC clone in combination with a third deletion of 7.5 Mb relative to the reference human genome. The break point for this third deletion is located within the LCR16a element that already forms part of rearrangement B.
Several of these rearrangements affect genes: break points B and C cause disruptions in copies of NOMO and PKD1 pseudogenes, respectively, and if the deletions in the BAC clones represent real deletions in the human genome, this would result in missing copies of a large number of genes (e.g., RRN3 in rearrangement A, RRN3, ABCC1, ABCC6, MYH11, XYLT1, SMG-1, CRYM and several more in rearrangement D). It therefore seems more probable that at least some of these rearrangements (e.g., rearrangement D) are in fact evidence for the formation of new LCR16a duplication clusters, and the observed deletions are due to the combination of duplication modules from different parts of chromosome 16. This is consistent with the observation that the region between 14.7 and 15.4 Mb and around 22.4 Mb on chromosome 16 has been indicated as being copy number variable in several studies (Locke et al. 2006; Redon et al. 2006; Marioni et al. 2007; Wong et al. 2007; Zogopoulos et al. 2007; Perry et al. 2008). Taken together, these data show that the regions duplicated by LCR16a are involved in recurrent and probably ongoing rearrangements.
LCR16a and Chromosomal Rearrangements in Gibbon
To further reinforce the evolutionary role of LCR16a-associated segmental duplications, we next wanted to know if they could also mediate gross chromosomal rearrangements, such as chromosomal translocations. We thought this possible because segmental duplications are frequently associated with chromosomal break points in primates and have been speculated to predispose to rearrangements, probably via nonallelic homologous recombination (Murphy et al. 2005). To answer if LCR16a-associated duplications could be involved in such a process, we focused on the gibbon or small ape (Hylobatidae) lineage, based on the rationale that the small apes underwent extensive chromosome reshuffling during evolution (Jauch et al. 1992; Murphy et al. 2001, 2003). Also, it was known that LCR16a had duplicated in gibbon (Johnson et al. 2001) and BAC coverage was sufficient for an in-depth sequence analysis. In a preliminary search, we found LCR16a-containing gibbon BAC clones from Kloss's gibbon (Hylobates klossii) and the white-cheeked gibbon (N. leucogenys), with the latter showing a higher coverage. We therefore selected N. leucogenys for subsequent investigations.
We compiled a list of N. leucogenys BAC clones that contained LCR16a sequence and identified a total of 15 clones that probably originated from five different regions of the gibbon genome. In some cases, the clones contained multiple copies of the LCR16a element and all the LCR16a sequences were localized within duplication clusters. Knowing that human LCR16a-associated duplication clusters have a modular structure, we inspected the gibbon duplications for similar properties. We found that similarly to the great apes, the duplication clusters in gibbon are composed of multiple modules. The modules are present in different order relative to each other in different duplication clusters, and among them LCR16a has the highest copy number. However, the modules we identified in gibbon are different from the LCR16a-associated modules observed in human (fig. 4). Indeed, even when regions of chromosome 16 are duplicated in man and gibbon alike (e.g., parts of the MPV17L or ABCC6 genes), the exact size and break points of the modules are different in the two species. This suggests that although the mechanism of duplication is the same in the two species (resulting in similar modular structure), LCR16a has mediated duplications on chromosome 16 independently in human and gibbon.
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Having established the presence of LCR16a-mediated duplication clusters in gibbon, we conducted further analysis to determine whether they were located within large-scale genome rearrangements. Thus, we compared our data with published gibbon chromosome rearrangements (Carbone et al. 2006
; Roberto et al. 2007) and found that both N. leucogenys chromosomes 7 and 18 contain syntenic segments corresponding to human chromosomes 4 and 5 adjacent to each other. Importantly, we observed LCR16a-associated duplication clusters in the syntenic breaks between these segments (fig. 4). Unfortunately, the information available is insufficient to establish whether the LCR16a-mediated duplications predated and contributed to these chromosomal rearrangements. However, our findings are consistent with the existing model for chromosome rearrangements and particularly suggestive in the light of our observation that LCR16a-associated duplications are also involved in structural variation in human. |
Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Existing Sequence Data and Disease-Causing Genes Are Useful Tools to Study Segmental Duplications
The aim of our study was to understand the consequences of recent segmental duplications. A major challenge for any such objective is that data from whole-genome surveys for these regions are often equivocal or they are not even included (Nguyen et al. 2006
). We therefore selected two partially duplicated Mendelian disorder genes, PKD1 and ABCC6 (fig. 1), because considerable effort has been made to develop mutation detection strategies that are specific for the functional copy. Thus, combining existing publicly available genomic, cDNA, and mutational data from PKD1 and ABCC6 provided us with a wealth of reliable information, without the ambiguities that are generally associated with whole-genome studies, demonstrating that Mendelian disorder genes are good models for studying recent segmental duplications.
Segmental Duplications: Catalysts of Genome Evolution
During our analysis of LCR16a-associated duplications, we found multiple partial copies of ABCC6 and PKD1 not only in human but also in chimpanzee and gibbon, showing that these regions are prone to duplication. We explained this based on the core duplicon hypothesis (supplementary file 1, Supplementary Material online), which states that the driving force for duplication is the association with an unstable (core) region.
At the same time, data from the literature (summarized in table 2) provide evidence from human and nonprimate species that the region surrounding ABCC6 is prone to rearrangements. Because the nonprimate rearrangements (table 2) occurred prior to LCR16a expansion, they must have been independent of LCR16a. This suggests that the ABCC6 locus is inherently unstable. It is therefore possible that LCR16a is not necessarily the initial cause for instability but it may preferentially associate with regions that are predisposed to rearrangements. However, regardless of the mechanism of duplication, once highly similar duplications are present in the genome, they have the potential to encode new genes and they enhance susceptibility for rearrangement (see sections below). Therefore, all duplicons, irrespective of their status as core or surrounding duplicon, will act as likely catalysts of primate genome evolution.
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Novel Genes Created by LCR16a-Mediated Duplication
Traditionally, the truncated copies of ABCC6 and PKD1 have been regarded as pseudogenes, with no physiological function, based on the fact that they could not encode a functional ABC transporter or polycystin protein, respectively. However, in the present study, we have identified several hominin-specific chimeric transcripts. Similar chimeric and in some cases, hominoid-specific genes have been previously described in association with recently duplicated regions (Bailey et al. 2002
; Bridgland et al. 2003; Paulding et al. 2003), and it is possible that such genes might have contributed to hominoid speciation. However, on an evolutionary timescale, only a small portion of segmental duplications lead to functional new genes. Thus, further studies will be necessary to resolve if the identified transcripts have any physiological role.
In the case of ABCC6-1, it is possible that the transcript is translated but it is not clear what the function—if there is any—of such a protein could be. While this manuscript was in preparation, Piehler et al. (2008) reported the ABCC6-1 transcript to show similar expression as the functional ABCC6 transcript. Interestingly, following targeted knockdown of the ABCC6-1 transcript, they also encountered significant reduction in ABCC6 expression, suggesting that the pseudogene transcript may have a function in regulating ABCC6 mRNA levels. A similar role has previously been established for the murine makorin-1 pseudogene (Hirotsune et al. 2003) and suggests that duplication of surrounding genomic regions has more functional relevance than just being a by-product of LCR16a duplication.
In the case of PKD1 pseudogenes, it is interesting that they form bipartite transcripts with copies of morpheus, the gene encoded by LCR16a. This is in line with the assumption that the increased copy number of "core genes" within diverse genomic environments was evolutionarily favorable. However, we established that all frames of the PKD1–morpheus fusion transcripts contain multiple termination codons, suggesting that they do not have protein-coding function. This is in agreement with findings by Bogdanova et al. (2001), who were unable to amplify pseudogene sequences in isolated polysome fractions, also suggesting that the pseudogenes are not translated. Notwithstanding, the transcribed exons of the PKD1 pseudogenes show a significantly higher divergence than the surrounding introns, which indicates a possible regulatory function. This is especially intriguing because the morpheus gene family is known for its strong positive selection in humans and African apes (Johnson et al. 2001).
Genetic Variability Is an Attribute of Segmental Duplications
We found that duplications associated with LCR16a are involved in gene conversion and rearrangements. Historically rearrangements have been classified as microscopic, if they are large enough to be identified using a microscope and submicroscopic, when they are beyond this resolution (1 kb–3 Mb in size) (Feuk et al. 2006). We have identified LCR16a-associated duplications to be involved in both types of rearrangements: chromosomal rearrangements in gibbon represent the former, structural variants in human the latter category. Because both phenomena, as well as gene conversion, can be explained by nonallelic homology–based recombination events, this suggests that the same mechanism can contribute to different levels of variability, potentially leading to different physiological outcomes.
Gene conversion causes sequence variation within a single gene. In the case of PKD1 and ABCC6, this can result in disease-causing alleles. However, most of the potential gene conversion events we detected were polymorphisms (70%; supplementary tables 3 and 4, Supplementary Material online), which suggest that gene conversion may contribute to the genetic variability of genes. This is contrary to the commonly accepted view that gene conversion events in which pseudogenes act as donors generally result only in loss-of-function mutations (Chen et al. 2007). Probably, this contradiction is due to a methodological bias because most known gene conversion events are anecdotal reports discovered in patients, whereas only a systematic study of mutations and polymorphisms can provide evidence that gene conversion could also be the cause for polymorphisms. Furthermore, this observation is in agreement with our finding that all previous observations of gene conversion were discovered during mutational analysis of patients. Our data are also in line with recent results showing that gene conversion plays a role in generating antibody diversity in humans (Cooper and Alder 2006; Darlow and Stott 2006; Pancer and Cooper 2006).
Chromosomal rearrangements affect several kilobases of sequence and are often associated with disease (Mazzarella and Schlessinger 1998; Emanuel and Shaikh 2001; Lupski and Stankiewicz 2005). It has also been suggested that rearranged chromosomes undergo accelerated evolution in parapatric populations, leading to a reduced recombination in heterokaryotypes (Navarro and Barton 2003; Marques-Bonet and Navarro 2005). If this is true, then LCR16a-mediated duplications could have favored divergence during primate evolution.
On a more everyday basis, our finding of multiple rearrangements affecting LCR16a-associated duplication clusters are important because they are consistent with previous results, which indicated that these regions are involved in fine-scale structural variation (Tuzun et al. 2005; Levy et al. 2007; Zogopoulos et al. 2007) and copy number variation ((Phakdeekitcharoen et al. 2000; Locke et al. 2006; Redon et al. 2006; Simon-Sanchez et al. 2007; Wong et al. 2007); (Marioni et al. 2007; Zogopoulos et al. 2007; Perry et al. 2008; Shen et al. 2008). Structural variations (i.e., larger polymorphisms, such as insertions, deletions, and inversions) have only recently been identified as a major source of human genetic variation but are already recognized as an area of interest by the National Human Genome Research Institute's Large-Scale Genome Sequencing Program (Eichler et al. 2007). However, a large number of genome-wide studies of structural variation have not reported any variants in this region (Sebat et al. 2004; Schulz et al. 2005; Sharp et al. 2005; Conrad et al. 2006; Goidts et al. 2006; McCarroll et al. 2006; Korbel et al. 2007; Mills et al. 2006); which probably reflects that the methods currently in use do not favor analysis of recently duplicated regions (Estivill and Armengol 2007; McCarroll and Altshuler 2007; Shen et al. 2008). This further shows that detailed investigations of single genes within segmental duplications are important to complement whole-genome studies.
In the future, further research will be necessary to understand how rearrangement/variation of this region influences phenotypic traits. Existing data suggest that affected features might be as diverse as autism, mental retardation (Ullmann et al. 2007), and multidrug resistance in cancer cells (O'Neill et al. 1998; Buys et al. 2007).
Innovation and Disease: An Evolutionary Trade-Off
Taken together, we find that the genomic regions associated with LCR16a have undergone independent duplications several times in primates and the segmental duplications created in this manner form a reservoir of "raw material" for new genes. At the same time, these duplications are substrates to nonallelic homologous recombination, which mainly results in genetic variation, both at the single base and structural level. However, these rearrangements also have the potential to cause disease. Therefore, the duplication of core regions and surrounding genomic fragments must be subject to an evolutionary trade-off over time, where manifestation of a given duplication will be dependent on how innovative traits are balanced by the capability of the duplications to cause disease.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary file 1 and tables 1–4 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The authors are grateful for the assistance of Hugues de Boussac, Gabriella Koblos, and Krisztina Fulop. The authors thank Peter Symmons and Noemi Lukacs for helpful discussions. Hungarian Scientific Research Fund (F-48684, NK-48729, NI-68950); Hungarian Comitee of Medical Sciences grant.
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Hervé Philippe, Associate Editor
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