Beiyuan Fu

Massive genomic rearrangement acquired in a single catastrophic event during cancer development

Summary Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%–3% of all cancers, across many subtypes, and is present in ∼25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer. PaperClip

Signatures of mutation and selection in the cancer genome

The cancer genome is moulded by the dual processes of somatic mutation and selection. Homozygous deletions in cancer genomes occur over recessive cancer genes, where they can confer selective growth advantage, and over fragile sites, where they are thought to reflect an increased local rate of DNA breakage. However, most homozygous deletions in cancer genomes are unexplained. Here we identified 2,428 somatic homozygous deletions in 746 cancer cell lines. These overlie 11% of protein-coding genes that, therefore, are not mandatory for survival of human cells. We derived structural signatures that distinguish between homozygous deletions over recessive cancer genes and fragile sites. Application to clusters of unexplained homozygous deletions suggests that many are in regions of inherent fragility, whereas a small subset overlies recessive cancer genes. The results illustrate how structural signatures can be used to distinguish between the influences of mutation and selection in cancer genomes. The extensive copy number, genotyping, sequence and expression data available for this large series of publicly available cancer cell lines renders them informative reagents for future studies of cancer biology and drug discovery.

Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer

Summary The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations. PaperClip

Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype

The Afrotheria, a supraordinal grouping of mammals whose radiation is rooted in Africa, is strongly supported by DNA sequence data but not by their disparate anatomical features. We have used flow-sorted human, aardvark, and African elephant chromosome painting probes and applied reciprocal painting schemes to representatives of two of the Afrotherian orders, the Tubulidentata (aardvark) and Proboscidea (elephants), in an attempt to shed additional light on the evolutionary affinities of this enigmatic group of mammals. Although we have not yet found any unique cytogenetic signatures that support the monophyly of the Afrotheria, embedded within the aardvark genome we find the strongest evidence yet of a mammalian ancestral karyotype comprising 2n = 44. This karyotype includes nine chromosomes that show complete conserved synteny to those of man, six that show conservation as single chromosome arms or blocks in the human karyotype but that occur on two different chromosomes in the ancestor, and seven neighbor-joining combinations (i.e., the synteny is maintained in the majority of species of the orders studied so far, but which corresponds to two chromosomes in humans). The comparative chromosome maps presented between human and these Afrotherian species provide further insight into mammalian genome organization and comparative genomic data for the Afrotheria, one of the four major evolutionary clades postulated for the Eutheria.

Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules

We have made a complete set of painting probes for the domestic horse by degenerate oligonucleotide-primed PCR amplification of flow-sorted horse chromosomes. The horse probes, together with a full set of those available for human, were hybridized onto metaphase chromosomes of human, horse and mule. Based on the hybridization results, we have generated genome-wide comparative chromosome maps involving the domestic horse, donkey and human. These maps define the overall distribution and boundaries of evolutionarily conserved chromosomal segments in the three genomes. Our results shed further light on the karyotypic relationships among these species and, in particular, the chromosomal rearrangements that underlie hybrid sterility and the occasional fertility of mules.

Estimation of rearrangement phylogeny for cancer genomes

Cancer genomes are complex, carrying thousands of somatic mutations including base substitutions, insertions and deletions, rearrangements, and copy number changes that have been acquired over decades. Recently, technologies have been introduced that allow generation of high-resolution, comprehensive catalogs of somatic alterations in cancer genomes. However, analyses of these data sets generally do not indicate the order in which mutations have occurred, or the resulting karyotype. Here, we introduce a mathematical framework that begins to address this problem. By using samples with accurate data sets, we can reconstruct relatively complex temporal sequences of rearrangements and provide an assembly of genomic segments into digital karyotypes. For cancer genes mutated in rearranged regions, this information can provide a chronological examination of the selective events that have taken place.

Transmissible Dog Cancer Genome Reveals the Origin and History of an Ancient Cell Lineage

Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy-number changes, and retrotransposon insertions. More than 10,000 genes carry nonsynonymous variants, and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived about 11,000 years ago. The cancer spawned by this individual dispersed across continents about 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden. An unusual tumor in dogs arose more than 10,000 years ago, and despite a huge mutational burden, its genome has remained stable.[Also see Perspective by Parker and Ostrander] Breaking Tumor Dogma Canine transmissible venereal tumor (CTVT) is an unusual form of cancer because the infectious agent is not a virus or bacterium but the tumor cells themselves, which are passed from one dog to another during coitus. To explore the molecular features of the tumor and its possible origins, Murchison et al. (p. 437; see the Perspective by Parker and Ostrander) sequenced the genomes of two CTVTs and their host dogs, one from Australia and one from Brazil. Although CTVT has acquired a massive number of genomic alterations, including hundreds of times more somatic mutations than are normally found in human cancers, the tumor cell genome has remained diploid and stable. Indeed, CTVT may first have arisen in a dog that lived more than 10,000 years ago.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice

Significance Assigning biological relevance and molecular function to large catalogues of mutated genes in cancer is a major challenge. Likewise, pinpointing drivers among thousands of transcriptionally or epigenetically dysregulated genes within a cancer is complex and limited by the lack of tools for high-throughput functional cancer genomic analyses. We show here for the first time, to our knowledge, application of the CRISPR/Cas9 genome engineering system for simultaneous (multiplexed) mutagenesis of large gene sets in adult mice, allowing high-throughput discovery and validation of cancer genes. We characterized applications of CRISPR/Cas9 multiplexing, resulting tumor phenotypes, and limitations of the methodology. By using defined genetic or environmental predisposing conditions, we also developed, to our knowledge, the first mouse models of CRISPR/Cas9-induced hepatocellular carcinoma and show how multiplexed CRISPR/Cas9 can facilitate functional genomic analyses of hepatobiliary cancers. Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

Cross-species chromosome painting in the golden mole and elephant-shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora

Cross–species painting (fluorescence in situ hybridization) with 23 (human Homo sapiens (HSA)) chromosome–specific painting probes (HSA 1–22 and the X) was used to delimit regions of homology on the chromosomes of the golden mole (Chrysochloris asiaticus) and elephant–shrew (Elephantulus rupestris). A cladistic interpretation of our data provides evidence of two unique associations, HSA 1/19p and 5/21/3, that support Afrotheria. The recognition of HSA 5/3/21 expands on the 3/21 synteny originally designated as an ancestral state for all eutherians. We have identified one adjacent segment combination (HSA2/8p/4) that is supportive of Afroinsectiphillia (aardvark, golden mole, elephant–shrew). Two segmental combinations (HSA 10q/17 and HSA 3/20) unite the aardvark and elephant–shrews as sister taxa. The finding that segmental syntenies in evolutionarily distant taxa can improve phylogenetic resolution suggests that they may be useful for testing sequence–based phylogenies of the early eutherian mammals. They may even suggest clades that sequence trees are not recovering with any consistency and thus encourage the search for additional rare genomic changes among afrotheres.

The genome phylogeny of domestic cat, red panda and five mustelid species revealed by comparative chromosome painting and G-banding.

Genome-wide homology maps among stone marten (Martes foina, 2n = 38), domestic cat (Felis catus, 2n = 38), American mink (Mustela vison, 2n = 30), yellow-throated marten (Martes flavigula, 2n = 40), Old World badger (Meles meles, 2n = 44), ferret badger (Melogale moschata, 2n = 38) and red panda (Ailurus fulgens, 2n = 36) have been established by cross-species chromosome painting with a complete set of stone marten probes. In total, 18 stone marten autosomal probes reveal 20, 19, 21, 18 and 21 pairs of homologous chromosomal segments in the respective genomes of American mink, yellow-throated marten, Old World badger, ferret badger and red panda. Reciprocal painting between stone marten and cat delineated 21 pairs of homologous segments shared in both stone marten and cat genomes. The chromosomal painting results indicate that most chromosomes of these species are highly conserved and show one-to-one correspondence with stone marten and cat chromosomes or chromosomal arms, and that only a few interchromosomal rearrangements (Robertsonian fusions and fissions) have occurred during species radiation. By comparing the distribution patterns of conserved chromosomal segments in both these species and the putative ancestral carnivore karyotype, we have reconstructed the pathway of karyotype evolution of these species from the putative 2n = 42 ancestral carnivore karyotype. Our results support a close phylogenetic relationship between the red panda and mustelids. The homology data presented in these maps will allow us to transfer the cat gene mapping data to other unmapped carnivore species.