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Colorectal carcinoma (CRC) is a common cause of mortality1, but a comprehensive description of its genomic landscape is lacking2-9. Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichia colipks+ colibactin in rectal cancers10 and the importance of the SBS93 signature11-13, which suggests that diet or smoking is a risk factor. Immune-escape driver mutations14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1), highlighting the role of whole-genome sequencing in optimizing patient care.
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BACKGROUND: Sarcomas are diverse neoplasms with highly variable histological appearances in which diagnosis is often challenging and management options for metastatic/unresectable disease limited. Many sarcomas have distinctive molecular alterations, but the range of alterations is large, variable in type and rapidly increasing, meaning that testing by limited panels is unable to capture the broad spectrum of clinically pertinent genomic drivers required. Paired whole genome sequencing (WGS) in contrast allows comprehensive assessment of small variants, copy number and structural variants along with mutational signature analysis and germline testing. METHODS: Introduction of WGS as a diagnostic standard for all eligible patients with known or suspected soft tissue sarcoma over a 2-year period at a soft tissue sarcoma treatment centre. RESULTS: WGS resulted in a refinement in the diagnosis in 37% of cases, identification of a target for personalised therapy in 33% of cases, and a germline alteration in 4% of cases. CONCLUSION: Introduction of WGS poses logistical and training challenges, but offers significant benefits to this group of patients.
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The 100 000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent in situ hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.
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Leucemia Linfocítica Crônica de Células B/genética , Sequenciamento Completo do Genoma/normas , Adulto , Idoso , Variações do Número de Cópias de DNA/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanism: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analysing the three-dimensional structures of protein-DNA complexes, here we show that the binding of arginine residues to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a prominent example of this effect. Minor-groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings indicate that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA-binding specificity.
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Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Sequência Rica em At/genética , Animais , Arginina/metabolismo , Sequência de Bases , DNA/genética , Proteínas de Ligação a DNA/química , Bases de Dados Factuais , Ligação de Hidrogênio , Lisina , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae , Eletricidade EstáticaRESUMO
The unexpected contamination of normal samples with tumour cells reduces variant detection sensitivity, compromising downstream analyses in canonical tumour-normal analyses. Leveraging whole-genome sequencing data available at Genomics England, we develop a tool for normal sample contamination assessment, which we validate in silico and against minimal residual disease testing. From a systematic review of [Formula: see text] patients with haematological malignancies and sarcomas, we find contamination across a range of cancer clinical indications and DNA sources, with highest prevalence in saliva samples from acute myeloid leukaemia patients, and sorted CD3+ T-cells from myeloproliferative neoplasms. Further exploration reveals 108 hotspot mutations in genes associated with haematological cancers at risk of being subtracted by standard variant calling pipelines. Our work highlights the importance of contamination assessment for accurate somatic variants detection in research and clinical settings, especially with large-scale sequencing projects being utilised to deliver accurate data from which to make clinical decisions for patient care.
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Neoplasias , Sequenciamento Completo do Genoma , Humanos , Genômica , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologiaRESUMO
Copy number alterations (CNAs) are among the most important genetic events in cancer, but their detection from sequencing data is challenging because of unknown sample purity, tumor ploidy, and general intra-tumor heterogeneity. Here, we present CNAqc, an evolution-inspired method to perform the computational validation of clonal and subclonal CNAs detected from bulk DNA sequencing. CNAqc is validated using single-cell data and simulations, is applied to over 4000 TCGA and PCAWG samples, and is incorporated into the validation process for the clinically accredited bioinformatics pipeline at Genomics England. CNAqc is designed to support automated quality control procedures for tumor somatic data validation.
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Variações do Número de Cópias de DNA , Neoplasias , Humanos , Algoritmos , Polimorfismo de Nucleotídeo Único , Neoplasias/genética , Neoplasias/patologia , Genômica/métodos , Biologia Computacional/métodosRESUMO
The Cancer Programme of the 100,000 Genomes Project was an initiative to provide whole-genome sequencing (WGS) for patients with cancer, evaluating opportunities for precision cancer care within the UK National Healthcare System (NHS). Genomics England, alongside NHS England, analyzed WGS data from 13,880 solid tumors spanning 33 cancer types, integrating genomic data with real-world treatment and outcome data, within a secure Research Environment. Incidence of somatic mutations in genes recommended for standard-of-care testing varied across cancer types. For instance, in glioblastoma multiforme, small variants were present in 94% of cases and copy number aberrations in at least one gene in 58% of cases, while sarcoma demonstrated the highest occurrence of actionable structural variants (13%). Homologous recombination deficiency was identified in 40% of high-grade serous ovarian cancer cases with 30% linked to pathogenic germline variants, highlighting the value of combined somatic and germline analysis. The linkage of WGS and longitudinal life course clinical data allowed the assessment of treatment outcomes for patients stratified according to pangenomic markers. Our findings demonstrate the utility of linking genomic and real-world clinical data to enable survival analysis to identify cancer genes that affect prognosis and advance our understanding of how cancer genomics impacts patient outcomes.
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Glioblastoma , Medicina de Precisão , Humanos , Genômica , Oncogenes , Mutação em Linhagem Germinativa/genéticaRESUMO
The discovery of cis-regulatory elements is a challenging problem in bioinformatics, owing to distal locations and context-specific roles of these elements in controlling gene regulation. Here we review the current bioinformatics methodologies and resources available for systematic discovery of cis-acting regulatory elements and conserved transcription factor binding sites in the chick genome. In addition, we propose and make available, a novel workflow using computational tools that integrate CTCF analysis to predict putative insulator elements, enhancer prediction, and TFBS analysis. To demonstrate the usefulness of this computational workflow, we then use it to analyze the locus of the gene Sox2 whose developmental expression is known to be controlled by a complex array of cis-acting regulatory elements. The workflow accurately predicts most of the experimentally verified elements along with some that have not yet been discovered. A web version of the CTCF tool, together with instructions for using the workflow can be accessed from http://toolshed.g2.bx.psu.edu/view/mkhan1980/ctcf_analysis. For local installation of the tool, relevant Perl scripts and instructions are provided in the directory named "code" in the supplementary materials.
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Galinhas/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genoma , Animais , Bases de Dados de Ácidos Nucleicos , Genômica/métodos , Internet , Software , Fluxo de TrabalhoRESUMO
Setting up the body plan during embryonic development requires the coordinated action of many signals and transcriptional regulators in a precise temporal sequence and spatial pattern. The last decades have seen an explosion of information describing the molecular control of many developmental processes. The next challenge is to integrate this information into logic "wiring diagrams" that visualize gene actions and outputs, have predictive power and point to key control nodes. Here, we provide an experimental workflow on how to construct gene regulatory networks using the chick as model system.
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Galinhas/genética , Redes Reguladoras de Genes , Animais , Embrião de Galinha , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação da Expressão GênicaRESUMO
The largest whole genome sequencing (WGS) endeavour involving cancer and rare diseases was initiated in the UK in 2015 and ran for 5 years. Despite its rarity, sarcoma ranked third overall among the number of patients' samples sent for sequencing. Herein, we recount the lessons learned by a specialist sarcoma centre that recruited close to 1000 patients to the project, so that we and others may learn from our experience. WGS data was generated from 597 patients, but samples from the remaining approximately 400 patients were not sequenced. This was largely accounted for by unsuitability due to extensive necrosis, secondary to neoadjuvant radiotherapy or chemotherapy, or being placed in formalin. The number of informative genomes produced was reduced further by a PCR amplification step. We showed that this loss of genomic data could be mitigated by sequencing whole genomes from needle core biopsies. Storage of resection specimens at 4 °C for up to 96 h overcame the challenge of freezing tissue out of hours including weekends. Removing access to formalin increased compliance to these storage arrangements. With over 70 different sarcoma subtypes described, WGS was a useful tool for refining diagnoses and identifying novel alterations. Genomes from 350 of the cohort of 597 patients were analysed in this study. Overall, diagnoses were modified for 3% of patients following review of the WGS findings. Continued refinement of the variant-calling bioinformatic pipelines is required as not all alterations were identified when validated against histology and standard of care diagnostic tests. Further research is necessary to evaluate the impact of germline mutations in patients with sarcoma, and sarcomas with evidence of hypermutation. Despite 50% of the WGS exhibiting domain 1 alterations, the number of patients with sarcoma who were eligible for clinical trials remains small, highlighting the need to revaluate clinical trial design.
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Biomarcadores Tumorais/genética , Mutação , Sarcoma/genética , Sequenciamento Completo do Genoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Análise Mutacional de DNA , Bases de Dados Genéticas , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Medicina de Precisão , Valor Preditivo dos Testes , Prognóstico , Projetos de Pesquisa , Sarcoma/mortalidade , Sarcoma/patologia , Sarcoma/terapia , Adulto JovemRESUMO
With the increasing number of eukaryotic genomes available, high-throughput automated tools for identification of regulatory DNA sequences are becoming increasingly feasible. Several computational approaches for the prediction of regulatory elements were recently developed. Here we combine the prediction of clusters of binding sites for transcription factors with context information taken from genome annotations. Target Explorer automates the entire process from the creation of a customized library of binding sites for known transcription factors through the prediction and annotation of putative target genes that are potentially regulated by these factors. It was specifically designed for the well-annotated Drosophila melanogaster genome, but most options can be used for sequences from other genomes as well. Target Explorer is available at http://trantor.bioc.columbia.edu/Target_Explorer/
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Drosophila melanogaster/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA/métodos , Software , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Genoma , Humanos , Internet , Interface Usuário-ComputadorRESUMO
Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.
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Proteínas de Fusão bcr-abl/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase/métodos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Neoplasia Residual , Medicina de Precisão/métodos , Sensibilidade e EspecificidadeRESUMO
Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncating mutation of the CPE gene (c.76_98del; p.E26RfsX68). Analysis detected no CPE expression in whole blood-derived RNA from the proband, consistent with nonsense-mediated decay. The morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotrophic hypogonadism seen in this individual recapitulates phenotypes in the previously described fat/fat and Cpe knockout mouse models, evidencing the importance of this peptide/hormone-processing enzyme in regulating body weight, metabolism, and brain and reproductive function in humans.
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Carboxipeptidase H/genética , Diabetes Mellitus Tipo 2/complicações , Deficiência Intelectual/complicações , Síndrome de Klinefelter/complicações , Mutação/genética , Obesidade Mórbida/complicações , Obesidade Mórbida/genética , Carboxipeptidase H/metabolismo , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Exoma/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Homozigoto , Humanos , Deficiência Intelectual/genética , Síndrome de Klinefelter/enzimologia , Síndrome de Klinefelter/genética , Masculino , Obesidade Mórbida/enzimologia , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
Embryonic polarity of invertebrates, amphibians and fish is specified largely by maternal determinants, which fixes cell fates early in development. In contrast, amniote embryos remain plastic and can form multiple individuals until gastrulation. How is their polarity determined? In the chick embryo, the earliest known factor is cVg1 (homologous to mammalian growth differentiation factor 1, GDF1), a transforming growth factor beta (TGFß) signal expressed posteriorly before gastrulation. A molecular screen to find upstream regulators of cVg1 in normal embryos and in embryos manipulated to form twins now uncovers the transcription factor Pitx2 as a candidate. We show that Pitx2 is essential for axis formation, and that it acts as a direct regulator of cVg1 expression by binding to enhancers within neighbouring genes. Pitx2, Vg1/GDF1 and Nodal are also key actors in left-right asymmetry, suggesting that the same ancient polarity determination mechanism has been co-opted to different functions during evolution.
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Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/metabolismo , Tamanho da Ninhada de Vivíparos/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha , Imunoprecipitação da Cromatina , Biologia Computacional , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Hibridização In Situ , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/genética , Fatores de Tempo , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
The functional annotation of the nonprotein-coding DNA of eukaryotic genomes is a problem of central importance. Phylogenetic footprinting methods, which attempt to identify functional regulatory regions by comparing orthologous genomic sequences of evolutionarily related species, have shown promising results. The main advantage of this class of approaches is that they do not require any knowledge of the regulating transcription factors. Here we describe a method called Enhancer Detection using only Genomic Information (EDGI), which integrates a traditional motif-discovery algorithm with a local permutation-clustering algorithm. Together, they can identify large regulatory elements (e.g., enhancers) as evolutionarily conserved order-independent clusters of short conserved motifs. We show that EDGI can distinguish between established sets of known enhancers and nonenhancers with 88% accuracy, rivaling predictions by methods that rely on the knowledge of the regulating transcription factors and their DNA-binding specificities. We tested EDGI's performance on a set of Drosophila genomes. Our results demonstrate that comparative genomic analysis of multiple closely related species has substantial power to identify key functional elements without additional biological knowledge.
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Algoritmos , Pegada de DNA/métodos , Drosophila/genética , Elementos Facilitadores Genéticos/genética , Genômica/métodos , Animais , Estudos de Avaliação como Assunto , FilogeniaRESUMO
The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.
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DNA/química , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Conformação de Ácido Nucleico , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Ativação Enzimática , Fatores de Transcrição Forkhead , Histidina/metabolismo , Proteínas de Homeodomínio/química , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Eletricidade Estática , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismoRESUMO
We show that reducing the activity of the Drosophila Runx protein Lozenge (Lz) during pupal development causes a decrease in cell death in the eye. We identified Lz-binding sites in introns of argos (aos) and klumpfuss (klu) and demonstrate that these genes are directly activated targets of Lz. Loss of either aos or klu reduces cell death, suggesting that Lz promotes apoptosis at least in part by regulating aos and klu. These results provide novel insights into the control of programmed cell death (PCD) by Lz during Drosophila eye development.