Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases

Despite the recent discoveries of and interest in numerous structural variations (SVs)--which include duplications and inversions--in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms. Here, we present a novel method of inducing duplications and inversions in a targeted manner without pre-manipulation of the genome. We found that zinc finger nucleases (ZFNs) designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted duplications and inversions of the genomic segments of up to a mega base pair (bp) in length between the two sites. Furthermore, we demonstrated that a ZFN pair could induce the inversion of a 140-kbp chromosomal segment that contains a portion of the blood coagulation factor VIII gene to mimic the inversion genotype that is associated with some cases of severe hemophilia A. This same ZFN pair could be used, in theory, to revert the inverted region to restore genomic integrity in these hemophilia A patients. We propose that ZFNs can be employed as molecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles. In addition, our method raises the possibility of correcting genetic defects caused by chromosomal rearrangements and holds new promise in gene and cell therapy.     

Mate pair sequencing for the detection of chromosomal aberrations in patients with intellectual disability and congenital malformations

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14–18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.     

Low-copy repeats at the human VIPR2 gene predispose to recurrent and nonrecurrent rearrangements

Submicroscopic structural variations, including deletions, duplications, inversions and more complex rearrangements, are widespread in normal human genomes. Inverted segmental duplications or highly identical low-copy repeat (LCR) sequences can mediate the formation of inversions and more complex structural rearrangements through non-allelic homologous recombination. In a patient with 7q36 inverted duplication/terminal deletion, we demonstrated the central role of a pair of short inverted LCRs in the vasoactive intestinal peptide receptor gene (VIPR2)-LCRs in generating the rearrangement. We also revealed a relatively common VIPR2-LCR-associated inversion polymorphism disrupting the gene in almost 1% of healthy subjects, and a small number of complex duplications/triplications. In genome-wide studies of several thousand patients, a significant association of rare microduplications with variable size, all involving VIPR2, with schizophrenia was recently described, suggesting that altered vasoactive intestinal peptide signaling is likely implicated in the pathogenesis of schizophrenia. Genetic testing for VIPR2-LCR-associated inversions should be performed on available cohorts of psychiatric patients to evaluate their potential pathogenic role.     

Genome-wide mapping and assembly of structural variant breakpoints in the mouse genome

Structural variation (SV) is a rich source of genetic diversity in mammals, but due to the challenges associated with mapping SV in complex genomes, basic questions regarding their genomic distribution and mechanistic origins remain unanswered. We have developed an algorithm (HYDRA) to localize SV breakpoints by paired-end mapping, and a general approach for the genome-wide assembly and interpretation of breakpoint sequences. We applied these methods to two inbred mouse strains: C57BL/6J and DBA/2J. We demonstrate that HYDRA accurately maps diverse classes of SV, including those involving repetitive elements such as transposons and segmental duplications; however, our analysis of the C57BL/6J reference strain shows that incomplete reference genome assemblies are a major source of noise. We report 7196 SVs between the two strains, more than two-thirds of which are due to transposon insertions. Of the remainder, 59% are deletions (relative to the reference), 26% are insertions of unlinked DNA, 9% are tandem duplications, and 6% are inversions. To investigate the origins of SV, we characterized 3316 breakpoint sequences at single-nucleotide resolution. We find that ∼16% of non-transposon SVs have complex breakpoint patterns consistent with template switching during DNA replication or repair, and that this process appears to preferentially generate certain classes of complex variants. Moreover, we find that SVs are significantly enriched in regions of segmental duplication, but that this effect is largely independent of DNA sequence homology and thus cannot be explained by non-allelic homologous recombination (NAHR) alone. This result suggests that the genetic instability of such regions is often the cause rather than the consequence of duplicated genomic architecture.In the six years since the first genome-wide analyses revealed extensive DNA copy number variation (CNV) among human individuals (Iafrate et al. 2004; Sebat et al. 2004), numerous studies have extended this observation in scope and scale with increasingly powerful genomic tools. It is now widely recognized that structural variation (SV), which includes duplications, deletions, inversions, transpositions, and other genomic rearrangements, is an abundant and functionally important class of genetic variation in mammals (Zhang et al. 2009a). Besides the emerging role of inherited variants in complex disease, new structural mutations contribute to sporadic human disorders, are a hallmark of tumor genomes, and drive the evolution of genes and species. For all of these reasons, it is important to generate accurate SV maps in many different organisms and cellular contexts, so that the biological consequences of SV may be assessed, and so that the molecular mechanisms that generate new variation may be fully understood.Several technical challenges have precluded a more complete understanding of the patterns and origins of SV. First, most studies have used array comparative genome hybridization (aCGH), which has limited resolution, cannot detect balanced rearrangements or reconstruct locus architecture, and has limited ability to detect SVs composed of multi-copy elements such as segmental duplications (SDs) or transposable elements (TEs). Second, sequence-based methods such as paired-end mapping (PEM) have emerged as a potent alternative to aCGH (Raphael et al. 2003; Tuzun et al. 2005; Korbel et al. 2007; Lee et al. 2008), but their practical utility has been limited by the high cost of “long-read” sequencing, and the computational difficulties associated with interpreting “short-read” sequence data from complex genomes. Thus, while a number of PEM-based algorithms have been developed to identify SV from short-read sequence data (Chen et al. 2009; Hormozdiari et al. 2009; Korbel et al. 2009; Medvedev et al. 2009) and newer methods have been devised to map SVs at higher resolution (Lee et al. 2009; Sindi et al. 2009), all short-read PEM studies except one (Hormozdiari et al. 2009) have restricted their analyses to paired-end reads that map uniquely to the reference genome. This approach is not ideal given that SVs often involve repeated sequences such as segmental duplications and transposons. Finally, it has been difficult to evaluate structural mutation mechanisms in an unbiased way because genome-wide studies have thus far characterized relatively few breakpoints at single-nucleotide resolution (Korbel et al. 2007; Kidd et al. 2008; Kim et al. 2008; Perry et al. 2008), and the relative contribution of different molecular mechanisms remains a matter of debate.Despite rapid advances in DNA sequencing technologies, affordable and accurate assembly of entire mammalian genomes remains years away. Indeed, even traditional methods have difficulty resolving complex genomic regions. In the interim, we argue that the optimal solution for breakpoint detection is a hybrid approach that combines PEM and local de novo assembly. Here we describe a general approach for unbiased detection, assembly, and mechanistic interpretation of SV breakpoints using both short and long reads, and apply it to whole-genome sequence data from two widely used inbred mouse strains. We show that our algorithms accurately identify diverse classes of SV, capture an unprecedented number of variants, and reveal novel breakpoint features. Of mechanistic significance, we report an abundance of complex SVs that appear to be derived from template switching during DNA replication or repair, and a propensity for duplicated genomic regions to generate new variants through mechanism(s) other than non-allelic homologous recombination (NAHR). A unique strength of this study is our choice of the mouse genome; because the reference genome is derived from an established inbred line (C57BL/6J), we were able to sequence an animal whose genome should be essentially identical to the reference. This important methodological control, which has not been present in any other PEM study, allowed us to distinguish true genetic variation from technical “noise” and poorly assembled genomic regions.     

Understanding the recent evolution of the human genome: insights from human-chimpanzee genome comparisons

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.     

Genome-Scale Evolution: Reconstructing Gene Orders in the Ancestral Species

Recent progress in genome-scale sequencing and comparative mapping raises new challenges in studies of genome rearrangements. Although the pairwise genome rearrangement problem is well-studied, algorithms for reconstructing rearrangement scenarios for multiple species are in great need. The previous approaches to multiple genome rearrangement problem were largely based on the breakpoint distance rather than on a more biologically accurate rearrangement (reversal) distance. Another shortcoming of the existing software tools is their inability to analyze rearrangements (inversions, translocations, fusions, and fissions) of multichromosomal genomes. This paper proposes a new multiple genome rearrangement algorithm that is based on the rearrangement (rather than breakpoint) distance and that is applicable to both unichromosomal and multichromosomal genomes. We further apply this algorithm for genome-scale phylogenetic tree reconstruction and deriving ancestral gene orders. In particular, our analysis suggests a new improved rearrangement scenario for a very difficult Campanulaceae cpDNA dataset and a putative rearrangement scenario for human, mouse and cat genomes.     

Evolutionary mechanisms shaping the genomic structure of the Williams-Beuren syndrome chromosomal region at human 7q11.23

About 5% of the human genome consists of segmental duplications or low-copy repeats, which are large, highly homologous (>95%) fragments of sequence. It has been estimated that these segmental duplications emerged during the past approximately 35 million years (Myr) of human evolution and that they correlate with chromosomal rearrangements. Williams-Beuren syndrome (WBS) is a segmental aneusomy syndrome that is the result of a frequent de novo deletion at 7q11.23, mediated by large (approximately 400-kb) region-specific complex segmental duplications composed of different blocks. We have precisely defined the structure of the segmental duplications on human 7q11.23 and characterized the copy number and structure of the orthologous regions in other primates (macaque, orangutan, gorilla, and chimpanzee). Our data indicate a recent origin and rapid evolution of the 7q11.23 segmental duplications, starting before the diversification of hominoids (approximately 12-16 million years ago [Mya]), with species-specific duplications and intrachromosomal rearrangements that lead to significant differences among those genomes. Alu sequences are located at most edges of the large hominoid-specific segmental duplications, suggesting that they might have facilitated evolutionary rearrangements. We propose a mechanistic model based on Alu-mediated duplicated transposition along with nonallelic homologous recombination for the generation and local expansion of the segmental duplications. The extraordinary rate of evolutionary turnover of this region, rich in segmental duplications, results in important genomic variation among hominoid species, which could be of functional relevance and predispose to disease.     

Transflip mutations produce deletions in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single‐nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine‐mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as “TransFlip” mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non‐recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non‐reciprocal, and non‐recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. © 2015 Wiley Periodicals, Inc.     

Catastrophic cellular events leading to complex chromosomal rearrangements in the germline

Although complex chromosomal rearrangements were thought to reflect the accumulation of DNA damage over time, recent studies have shown that such rearrangements frequently arise from ‘all‐at‐once’ catastrophic cellular events. These events, designated chromothripsis, chromoanasynthesis, and chromoanagenesis, were first documented in the cancer genome and subsequently observed in the germline. These events likely result from micronucleus‐mediated chromosomal shattering and subsequent random reassembly of DNA fragments, although several other mechanisms have also been proposed. Typically, only one or a few chromosomes of paternal origin are affected per event. These events can produce intrachromosomal deletions, duplications, inversions, and translocations, as well as interchromosomal translocations. Germline complex rearrangements of autosomes often result in developmental delay and dysmorphic features, whereas X chromosomal rearrangements are usually associated with relatively mild clinical manifestations. The concept of these catastrophic events provides novel insights into the etiology of human genomic disorders. This review introduces the molecular characteristics and phenotypic outcomes of catastrophic cellular events in the germline.     

Structure of chromosomal duplicons and their role in mediating human genomic disorders

Chromosome-specific low-copy repeats, or duplicons, occur in multiple regions of the human genome. Homologous recombination between different duplicon copies leads to chromosomal rearrangements, such as deletions, duplications, inversions, and inverted duplications, depending on the orientation of the recombining duplicons. When such rearrangements cause dosage imbalance of a developmentally important gene(s), genetic diseases now termed genomic disorders result, at a frequency of 0.7-1/1000 births. Duplicons can have simple or very complex structures, with variation in copy number from 2 to >10 repeats, and each varying in size from a few kilobases in length to hundreds of kilobases. Analysis of the different duplicons involved in human genomic disorders identifies features that may predispose to recombination, including large size and high sequence identity between the recombining copies, putative recombination promoting features, and the presence of multiple genes/pseudogenes that may include genes expressed in germ cells. Most of the chromosome rearrangements involve duplicons near pericentromeric regions, which may relate to the propensity of such regions to accumulate duplicons. Detailed analyses of the structure, polymorphic variation, and mechanisms of recombination in genomic disorders, as well as the evolutionary origin of various duplicons will further our understanding of the structure, function, and fluidity of the human genome.     

The automatic detection of homologous regions (ADHoRe) and its application to microcolinearity between Arabidopsis and rice

It is expected that one of the merits of comparative genomics lies in the transfer of structural and functional information from one genome to another. This is based on the observation that, although the number of chromosomal rearrangements that occur in genomes is extensive, different species still exhibit a certain degree of conservation regarding gene content and gene order. It is in this respect that we have developed a new software tool for the Automatic Detection of Homologous Regions (ADHoRe). ADHoRe was primarily developed to find large regions of microcolinearity, taking into account different types of microrearrangements such as tandem duplications, gene loss and translocations, and inversions. Such rearrangements often complicate the detection of colinearity, in particular when comparing more anciently diverged species. Application of ADHoRe to the complete genome of Arabidopsis and a large collection of concatenated rice BACs yields more than 20 regions showing statistically significant microcolinearity between both plant species. These regions comprise from 4 up to 11 conserved homologous gene pairs. We predict the number of homologous regions and the extent of microcolinearity to increase significantly once better annotations of the rice genome become available.     

Fourfold faster rate of genome rearrangement in nematodes than in Drosophila

We compared the genome of the nematode Caenorhabditis elegans to 13% of that of Caenorhabditis briggsae, identifying 252 conserved segments along their chromosomes. We detected 517 chromosomal rearrangements, with the ratio of translocations to inversions to transpositions being approximately 1:1:2. We estimate that the species diverged 50-120 million years ago, and that since then there have been 4030 rearrangements between their whole genomes. Our estimate of the rearrangement rate, 0.4-1.0 chromosomal breakages/Mb per Myr, is at least four times that of Drosophila, which was previously reported to be the fastest rate among eukaryotes. The breakpoints of translocations are strongly associated with dispersed repeats and gene family members in the C. elegans genome.     

Remembrance of things past retrieved from the Paramecium genome

Paramecium and other ciliates are the only unicellular eukaryotes that separate germinal and somatic functions. A germline micronucleus transmits the genetic information to sexual progeny, while a somatic macronucleus expresses the genetic information during vegetative growth to determine the phenotype. At each sexual generation, a new macronucleus develops from the zygotic nucleus through programmed rearrangements of the germline genome. Paramecium tetraurelia somatic genome sequencing, reviewed here, has provided insight into the organization and evolution of the genome. A series of at least 3 whole genome duplications was detected in the Paramecium lineage and selective pressures that determine the fate of the gene duplicates analyzed. Variability in the somatic DNA was characterized and could be attributed to the genome rearrangement processes. Since, in Paramecium, alternative genome rearrangement patterns can be inherited across sexual generations by homology-dependent epigenetic mechanisms and can affect phenotype, I discuss the possibility that ciliate nuclear dimorphism buffers genetic variation hidden in the germline.     

Segmental duplication density decrease with distance to human-mouse breaks of synteny

Segmental duplications are large genomic segments of recent origin and nearly identical sequence. Segmental duplications account for up to 5% of the human genome and they are often involved in genomic rearrangements and human disease. We developed a rapid computational method to characterize segmental duplications in the mouse and the human genomes according to four sequence assemblies for each species. Segmental duplication content in the mouse genome assemblies has largely changed over the four releases (from 0.2 to 1.2%, 4.5 and 3.0%), while in the four human assemblies duplication content was 4.8, 3.5, 3.7 and 3.7%, respectively. This suggests that cataloguing and assembling duplications has been challenging in both genomes and any interpretation of comparative analyses of duplication content must keep this in perspective to avoid artifacts. Human and mouse segmental duplications are more frequent than expected in regions where there is a syntenic discontinuity and the duplication content in syntenic regions decreases significantly with distance from breakpoints of synteny. These observations indicate that in mouse and human the frequency of segmental duplications is strongly correlated with distance to human and mouse syntenic breaks or the most dynamic regions in evolution..     

Segmental duplications flank the multiple sclerosis locus on chromosome 17q

Large chromosomal rearrangements, duplications, and inversions are relatively common in mammalian genomes. Here we report interesting features of DNA strands flanking a Multiple Sclerosis (MS) susceptibility locus on Chromosome 17q24. During the positional cloning process of this 3-Mb locus, several markers showed a radiation hybrid clone retention rate above the average (1.8-fold), suggestive for the existence of duplicated sequences in this region. FISH studies demonstrated multiple signals with three of the tested regional BACs, and 24 BACs out of 187 showed evidence for duplication in shotgun sequence comparisons of the 17q22-q24 region. Specifically, the MS haplotype region proved to be flanked by palindromic sequence stretches and by long segmental intrachromosomal duplications in which highly homologous DNA sequences (>96% identity) are present at both ends of the haplotype. Moreover, the 3-Mb DNA segment, flanked by the duplications, is inverted in the mouse genome when compared with the orientation in human and chimp. The segmental duplication architecture surrounding the MS locus raises the possibility that a nonallelic homologous recombination between duplications could affect the biological activity of the regional genes, perhaps even contributing to the genetic background of MS.     

Enredo and Pecan: genome-wide mammalian consistency-based multiple alignment with paralogs

Pairwise whole-genome alignment involves the creation of a homology map, capable of performing a near complete transformation of one genome into another. For multiple genomes this problem is generalized to finding a set of consistent homology maps for converting each genome in the set of aligned genomes into any of the others. The problem can be divided into two principal stages. First, the partitioning of the input genomes into a set of colinear segments, a process which essentially deals with the complex processes of rearrangement. Second, the generation of a base pair level alignment map for each colinear segment. We have developed a new genome-wide segmentation program, Enredo, which produces colinear segments from extant genomes handling rearrangements, including duplications. We have then applied the new alignment program Pecan, which makes the consistency alignment methodology practical at a large scale, to create a new set of genome-wide mammalian alignments. We test both Enredo and Pecan using novel and existing assessment analyses that incorporate both real biological data and simulations, and show that both independently and in combination they outperform existing programs. Alignments from our pipeline are publicly available within the Ensembl genome browser.