Fengtang Yang

Global variation in copy number in the human genome

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

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.

A comparative study of karyotypes of muntjacs by chromosome painting

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific “paints” from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4′, 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.

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

Copy number variation and evolution in humans and chimpanzees

Copy number variants (CNVs) underlie many aspects of human phenotypic diversity and provide the raw material for gene duplication and gene family expansion. However, our understanding of their evolutionary significance remains limited. We performed comparative genomic hybridization on a single human microarray platform to identify CNVs among the genomes of 30 humans and 30 chimpanzees as well as fixed copy number differences between species. We found that human and chimpanzee CNVs occur in orthologous genomic regions far more often than expected by chance and are strongly associated with the presence of highly homologous intrachromosomal segmental duplications. By adapting population genetic analyses for use with copy number data, we identified functional categories of genes that have likely evolved under purifying or positive selection for copy number changes. In particular, duplications and deletions of genes with inflammatory response and cell proliferation functions may have been fixed by positive selection and involved in the adaptive phenotypic differentiation of humans and chimpanzees.

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.

Adaptive Evolution of UGT2B17 Copy-Number Variation

The human UGT2B17 gene varies in copy number from zero to two per individual and also differs in mean number between populations from Africa, Europe, and East Asia. We show that such a high degree of geographical variation is unusual and investigate its evolutionary history. This required first reinterpreting the reference sequence in this region of the genome, which is misassembled from the two different alleles separated by an artifactual gap. A corrected assembly identifies the polymorphism as a 117 kb deletion arising by nonallelic homologous recombination between approximately 4.9 kb segmental duplications and allows the deletion breakpoint to be identified. We resequenced approximately 12 kb of DNA spanning the breakpoint in 91 humans from three HapMap and one extended HapMap populations and one chimpanzee. Diversity was unusually high and the time to the most recent common ancestor was estimated at approximately 2.4 or approximately 3.0 million years by two different methods, with evidence of balancing selection in Europe. In contrast, diversity was low in East Asia where a single haplotype predominated, suggesting positive selection for the deletion in this part of the world.

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10–20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5–6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.

A high utility integrated map of the pig genome

BackgroundThe domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.ResultsHere we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.ConclusionThe map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.