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

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.

The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes.

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.

Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.

The aorta after coarctation repair - effects of calibre and curvature on arterial haemodynamics.

BACKGROUND: Aortic shape has been proposed as an important determinant of adverse haemodynamics following coarctation repair. However, previous studies have not demonstrated a consistent relationship between shape and vascular load. In this study, 3D aortic shape was evaluated using principal component analysis (PCA), allowing investigation of the relationship between 3D shape and haemodynamics. METHODS: Sixty subjects (38 male, 25.0 ± 7.8 years) with repaired coarctation were recruited. Central aortic haemodynamics including wave intensity analysis were measured noninvasively using a combination of blood pressure and phase contrast cardiovascular magnetic resonance (CMR). 3D curvature and radius data were derived from CMR angiograms. PCA was separately performed on 3D radius and curvature data to assess the role of arch geometry on haemodynamics. Clinical findings were corroborated using 1D vascular models. RESULTS: There were no independent associations between 3D curvature and any hemodynamic parameters. However, the magnitude of the backwards compression wave was related to the 1st (r = - 0.36, p = 0.005), 3rd (r = 0.27, p = 0.036) and 4th (r = - 0.31, p = 0.017) principle components of radius. The 4th principle componentof radius also correlated with central aortic systolic pressure. These aortas had larger aortic roots, more transverse arch hypoplasia and narrower aortic isthmuses. CONCLUSIONS: There are major modes of variation in 3D aortic shape after coarctation repair witha modest association between variation in aortic radius and pathological wave reflections, but not with 3D curvature. Taken together, these data suggest that shape is not the major determinant of vascular load following coarctation repair, and calibre is more important than curvature.

Correction to: The aorta after coarctation repair - effects of calibre and curvature on arterial haemodynamics.

In the original version of this article [1], published on 11 April 2019, there is 1 error in the 'Conclusion' paragraph of the abstract.

The cardiovascular phenotype of childhood hypertension: a cardiac magnetic resonance study.

BACKGROUND: The cardiovascular phenotype is poorly characterized in treated pediatric hypertension. Cardiovascular magnetic resonance imaging (MRI) can be used to better characterize both cardiac and vascular phenotype in children with hypertension. OBJECTIVE: To use MRI to determine the cardiac and vascular phenotypes of different forms of treated hypertension and compare the results with those of healthy children. MATERIALS AND METHODS: Sixty children (15 with chronic renal disease with hypertension, 15 with renovascular hypertension, 15 with essential hypertension and 15 healthy subjects) underwent MRI with noninvasive blood pressure measurements. Cardiovascular parameters measured include systemic vascular resistance, total arterial compliance, left ventricular mass and volumetric data, ejection fraction and myocardial velocity. Between-group comparisons were used to investigate differences in the hypertension types. RESULTS: Renal hypertension was associated with elevated vascular resistance (P≤0.007) and normal arterial compliance. Conversely, children with essential hypertension had normal resistance but increased compliance (P=0.001). Renovascular hypertension was associated with both increased resistance and compliance (P≤0.03). There was no difference in ventricular volumes, mass or cardiac output between groups. Children with renal hypertension also had lower systolic and diastolic myocardial velocities. CONCLUSION: Cardiovascular MRI may identify distinct vascular and cardiac phenotypes in different forms of treated childhood hypertension. Future studies are needed to investigate how this may inform further optimisation of blood pressure treatment in different types of hypertension.

Copy number variation arising from gene conversion on the human Y chromosome.

We describe the variation in copy number of a ~ 10 kb region overlapping the long intergenic noncoding RNA (lincRNA) gene, TTTY22, within the IR3 inverted repeat on the short arm of the human Y chromosome, leading to individuals with 0-3 copies of this region in the general population. Variation of this CNV is common, with 266 individuals having 0 copies, 943 (including the reference sequence) having 1, 23 having 2 copies, and two having 3 copies, and was validated by breakpoint PCR, fibre-FISH, and 10× Genomics Chromium linked-read sequencing in subsets of 1234 individuals from the 1000 Genomes Project. Mapping the changes in copy number to the phylogeny of these Y chromosomes previously established by the Project identified at least 20 mutational events, and investigation of flanking paralogous sequence variants showed that the mutations involved flanking sequences in 18 of these, and could extend over > 30 kb of DNA. While either gene conversion or double crossover between misaligned sister chromatids could formally explain the 0-2 copy events, gene conversion is the more likely mechanism, and these events include the longest non-allelic gene conversion reported thus far. Chromosomes with three copies of this CNV have arisen just once in our data set via another mechanism: duplication of 420 kb that places the third copy 230 kb proximal to the existing proximal copy. Our results establish gene conversion as a previously under-appreciated mechanism of generating copy number changes in humans and reveal the exceptionally large size of the conversion events that can occur.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.

A comprehensive characterization of myocardial and vascular phenotype in pediatric chronic kidney disease using cardiovascular magnetic resonance imaging.

BACKGROUND: Children with chronic kidney disease (CKD) have increased cardiovascular mortality. Identifying high-risk children who may benefit from further therapeutic intervention is difficult as cardiovascular abnormalities are subtle. Although transthoracic echocardiography may be used to detect sub-clinical abnormalities, it has well-known problems with reproducibility that limit its ability to accurately detect these changes. Cardiovascular magnetic resonance (CMR) is the reference standard method for assessing blood flow, cardiac structure and function. Furthermore, recent innovations enable the assessment of radial and longitudinal myocardial velocity, such that detection of sub-clinical changes is now possible. Thus, CMR may be ideal for cardiovascular assessment in pediatric CKD. This study aims to comprehensively assess cardiovascular function in pediatric CKD using CMR and determine its relationship with CKD severity. METHODS: A total of 120 children (40 mild, 40 moderate, 20 severe pre-dialysis CKD subjects and 20 healthy controls) underwent CMR with non-invasive blood pressure (BP) measurements. Cardiovascular parameters measured included systemic vascular resistance (SVR), total arterial compliance (TAC), left ventricular (LV) structure, ejection fraction (EF), cardiac timings, radial and longitudinal systolic and diastolic myocardial velocities. Between group comparisons and regression modelling were used to identify abnormalities in CKD and determine the effects of renal severity on myocardial function. RESULTS: The elevation in mean BP in CKD was accompanied by significantly increased afterload (SVR), without evidence of arterial stiffness (TAC) or increased fluid overload. Left ventricular volumes and global function were not abnormal in CKD. However, there was evidence of LV remodelling, prolongation of isovolumic relaxation time and reduced systolic and diastolic myocardial velocities. CONCLUSION: Abnormal cardiovascular function is evident in pre-dialysis pediatric CKD. Novel CMR biomarkers may be useful for the detection of subtle abnormalities in this population. Further studies are needed to determine to prognostic value of these biomarkers.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

UNLABELLED: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. AVAILABILITY AND IMPLEMENTATION: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-Tradis CONTACT: parkhill@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PET-CMR in heart failure - synergistic or redundant imaging?

Imaging in heart failure (HF) provides data for diagnosis, prognosis and disease monitoring. Both MRI and nuclear imaging techniques have been successfully used for this purpose in HF. Positron Emission Tomography-Cardiac Magnetic Resonance (PET-CMR) is an example of a new multimodality diagnostic imaging technique with potential applications in HF. The threshold for adopting a new diagnostic tool to clinical practice must necessarily be high, lest they exacerbate costs without improving care. New modalities must demonstrate clinical superiority, or at least equivalence, combined with another important advantage, such as lower cost or improved patient safety. The purpose of this review is to outline the current status of multimodality PET-CMR with regard to HF applications, and determine whether the clinical utility of this new technology justifies the cost.

Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry.

Supergenes are tight clusters of loci that facilitate the co-segregation of adaptive variation, providing integrated control of complex adaptive phenotypes. Polymorphic supergenes, in which specific combinations of traits are maintained within a single population, were first described for 'pin' and 'thrum' floral types in Primula and Fagopyrum, but classic examples are also found in insect mimicry and snail morphology. Understanding the evolutionary mechanisms that generate these co-adapted gene sets, as well as the mode of limiting the production of unfit recombinant forms, remains a substantial challenge. Here we show that individual wing-pattern morphs in the polymorphic mimetic butterfly Heliconius numata are associated with different genomic rearrangements at the supergene locus P. These rearrangements tighten the genetic linkage between at least two colour-pattern loci that are known to recombine in closely related species, with complete suppression of recombination being observed in experimental crosses across a 400-kilobase interval containing at least 18 genes. In natural populations, notable patterns of linkage disequilibrium (LD) are observed across the entire P region. The resulting divergent haplotype clades and inversion breakpoints are found in complete association with wing-pattern morphs. Our results indicate that allelic combinations at known wing-patterning loci have become locked together in a polymorphic rearrangement at the P locus, forming a supergene that acts as a simple switch between complex adaptive phenotypes found in sympatry. These findings highlight how genomic rearrangements can have a central role in the coexistence of adaptive phenotypes involving several genes acting in concert, by locally limiting recombination and gene flow.

Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

BACKGROUND: The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. RESULTS: We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. CONCLUSION: We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

Genetic basis of Y-linked hearing impairment.

A single Mendelian trait has been mapped to the human Y chromosome: Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ∼160 kb of DNA from chromosome 1, in the pericentric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more genes from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.

The patterns and dynamics of genomic instability in metastatic pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97-98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites, and how the tumour disseminates. Here we harness advances in DNA sequencing to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2-M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

The comparative role of echocardiography and MRI for identifying critical lesions in patients with single-ventricle physiology, before bidirectional cavopulmonary connection.

BACKGROUND: In the era of multi-modality imaging, this study compared contemporary, pre-operative echocardiography and cardiac MRI in predicting the need for intervention on additional lesions before surgical bidirectional cavopulmonary connection. METHODS: A total of 72 patients undergoing bidirectional cavopulmonary connection for single-ventricle palliation between 2007 and 2012, who underwent pre-operative assessment using both echocardiography and MRI, were included. The pre-determined outcome measure was any additional surgical or catheter-based intervention within 6 months of bidirectional cavopulmonary connection. Indices assessed were as follows: indexed dimensions of right and left pulmonary arteries, coarctation of the aorta, adequacy of interatrial communication, and degree of atrioventricular valve regurgitation. RESULTS: Median age at bidirectional cavopulmonary connection was 160 days (interquartile range 121-284). The following MRI parameters predicted intervention: Z score for right pulmonary artery (odds ratio 1.77 (95% confidence interval 1.12-2.79, p=0.014)) and left pulmonary artery dimensions (odds ratio 1.45 (1.04-2.00, p=0.027)) and left pulmonary artery report conclusion (odds ratio 1.57 (1.06-2.33)). The magnetic resonance report predicted aortic arch intervention (odds ratio 11.5 (3.5-37.7, p=0.00006)). The need for atrioventricular valve repair was associated only with magnetic resonance regurgitation fraction score (odds ratio 22.4 (1.7-295.1, p=0.018)). Echocardiography assessment was superior to MRI for predicting intervention on interatrial septum (odds ratio 27.7 (6.3-121.6, p=0.00001)). CONCLUSION: For branch pulmonary arteries, aortic arch, and atrioventricular valve regurgitation, MRI parameters more reliably predict the need for intervention; however, echocardiography more accurately identified the adequacy of interatrial communication. Approaching bidirectional cavopulmonary connection, the diagnostic strengths of MRI and echocardiography should be acknowledged when considering intervention.

Genome variation and evolution of the malaria parasite Plasmodium falciparum.

Infections with the malaria parasite Plasmodium falciparum result in more than 1 million deaths each year worldwide. Deciphering the evolutionary history and genetic variation of P. falciparum is critical for understanding the evolution of drug resistance, identifying potential vaccine candidates and appreciating the effect of parasite variation on prevalence and severity of malaria in humans. Most studies of natural variation in P. falciparum have been either in depth over small genomic regions (up to the size of a small chromosome) or genome wide but only at low resolution. In an effort to complement these studies with genome-wide data, we undertook shotgun sequencing of a Ghanaian clinical isolate (with fivefold coverage), the IT laboratory isolate (with onefold coverage) and the chimpanzee parasite P. reichenowi (with twofold coverage). We compared these sequences with the fully sequenced P. falciparum 3D7 isolate genome. We describe the most salient features of P. falciparum polymorphism and adaptive evolution with relation to gene function, transcript and protein expression and cellular localization. This analysis uncovers the primary evolutionary changes that have occurred since the P. falciparum-P. reichenowi speciation and changes that are occurring within P. falciparum.