RESUMEN
Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.
Asunto(s)
Epigenómica , Enfermedades del Sistema Inmune/genética , Monocitos/metabolismo , Neutrófilos/metabolismo , Linfocitos T/metabolismo , Transcripción Genética , Adulto , Anciano , Empalme Alternativo , Femenino , Predisposición Genética a la Enfermedad , Células Madre Hematopoyéticas/metabolismo , Código de Histonas , Humanos , Masculino , Persona de Mediana Edad , Sitios de Carácter Cuantitativo , Adulto JovenRESUMEN
Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.
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Análisis Mutacional de ADN , Genoma Humano/genética , Meduloblastoma/clasificación , Meduloblastoma/genética , Secuenciación Completa del Genoma , Carcinogénesis/genética , Proteínas Portadoras/genética , Estudios de Cohortes , Metilación de ADN , Conjuntos de Datos como Asunto , Epistasis Genética , Genómica , Humanos , Terapia Molecular Dirigida , Proteínas Musculares/genética , Mutación , Oncogenes/genética , Factores de Transcripción/genética , Proteínas Wnt/genéticaRESUMEN
Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
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Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica/genética , Meduloblastoma/clasificación , Meduloblastoma/patología , Factores de Transcripción/metabolismo , Animales , Neoplasias Cerebelosas/clasificación , Femenino , Redes Reguladoras de Genes/genética , Genes Relacionados con las Neoplasias/genética , Genes Reporteros/genética , Humanos , Masculino , Meduloblastoma/genética , Ratones , Reproducibilidad de los Resultados , Pez Cebra/genéticaRESUMEN
Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
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Variación Genética/genética , Genoma Humano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Transcriptoma/genética , Alelos , Línea Celular Transformada , Exones/genética , Perfilación de la Expresión Génica , Humanos , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
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Neoplasias Cerebelosas/genética , Genoma Humano/genética , Meduloblastoma/genética , Envejecimiento/genética , Secuencia de Aminoácidos , Transformación Celular Neoplásica , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/diagnóstico , Neoplasias Cerebelosas/patología , Niño , Cromatina/metabolismo , Cromosomas Humanos/genética , ARN Helicasas DEAD-box/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Genómica , Proteínas Hedgehog/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Demetilasas/genética , Humanos , Meduloblastoma/clasificación , Meduloblastoma/diagnóstico , Meduloblastoma/patología , Metilación , Mutación/genética , Tasa de Mutación , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatasas/genética , Poliploidía , Receptores de Superficie Celular/genética , Análisis de Secuencia de ARN , Transducción de Señal , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N). RESULTS: We found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements. CONCLUSIONS: Parent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair ß-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074).
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Obesidad/genética , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Ácidos Grasos/metabolismo , Femenino , Hígado/metabolismo , Masculino , Ratones , Pubertad/genéticaRESUMEN
BACKGROUND: Gene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions. RESULTS: Using HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods. CONCLUSIONS: We could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.
Asunto(s)
ARN Mensajero/aislamiento & purificación , Perfilación de la Expresión Génica , Biblioteca de Genes , Células HEK293 , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Empalme del ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/aislamiento & purificación , ARN Mensajero/genética , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
MOTIVATION: Protocols to generate strand-specific transcriptomes with next-generation sequencing platforms have been used by the scientific community roughly since 2008. Strand-specific reads allow for detection of antisense events and a higher resolution of expression profiles enabling extension of current transcript annotations. However, applications making use of this strandedness information are still scarce. RESULTS: Here we present a tool (Janus), which focuses on the identification of transcriptional active regions in antisense orientation to known and novel transcribed elements of the genome. Janus can compare the antisense events of multiple samples and assigns scores to identify mutual expression of either transcript in a sense/antisense pair, which could hint to regulatory mechanisms. Janus is able to make use of single-nucleotide variant (SNV) and methylation data, if available, and reports the sense to antisense ratio of regions in the vicinity of the identified genetic and epigenetic variation. Janus interrogates positions of heterozygous SNVs to identify strand-specific allelic imbalance. AVAILABILITY: Janus is written in C/C++ and freely available at http://www.ikmb.uni-kiel.de/janus/janus.html under terms of GNU General Public License, for both, Linux and Windows 64×. Although the binaries will work without additional downloads, the software depends on bamtools (https://github.com/pezmaster31/bamtools) for compilation. A detailed tutorial section is included in the first section of the supplemental material and included as brief readme.txt in the tutorial archive. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , ARN sin Sentido/biosíntesis , Programas Informáticos , Línea Celular Transformada , Metilación de ADN , Variación Genética , HumanosRESUMEN
Preserving the original RNA orientation information in RNA-Sequencing (RNA-Seq) experiment is essential to the analysis and understanding of the complexity of mammalian transcriptomes. We describe herein a simple, robust, and time-effective protocol for generating strand-specific RNA-seq libraries suited for the Illumina sequencing platform. We modified the Illumina TruSeq RNA sample preparation by implementing the strand specificity feature using the dUTP method. This protocol uses low amounts of starting material and allows a fast processing within two days. It can be easily implemented and requires only few additional reagents to the original Illumina kit.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Análisis de Secuencia de ARN/métodos , Nucleótidos de Desoxiuracil/genética , Humanos , TranscriptomaRESUMEN
High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.
Asunto(s)
ADN Complementario/química , Perfilación de la Expresión Génica , Análisis de Secuencia de ADN/métodos , Animales , Nucleótidos de Desoxiuracil/metabolismo , Genes Fúngicos , Ratones , Regiones Promotoras Genéticas , ARN sin Sentido/biosíntesis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the Huntingtin gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, IFITM3, IFI6 and IRF7. Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.
RESUMEN
The chimeric transcription factor TCF3-HLF defines an incurable acute lymphoblastic leukemia subtype. Here we decipher the regulome of endogenous TCF3-HLF and dissect its essential transcriptional components and targets by functional genomics. We demonstrate that TCF3-HLF recruits HLF binding sites at hematopoietic stem cell/myeloid lineage associated (super-) enhancers to drive lineage identity and self-renewal. Among direct targets, hijacking an HLF binding site in a MYC enhancer cluster by TCF3-HLF activates a conserved MYC-driven transformation program crucial for leukemia propagation in vivo. TCF3-HLF pioneers the cooperation with ERG and recruits histone acetyltransferase p300 (EP300), conferring susceptibility to EP300 inhibition. Our study provides a framework for targeting driving transcriptional dependencies in this fatal leukemia.
Asunto(s)
Proteína p300 Asociada a E1A/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas de Unión al ADN/genética , Humanos , Translocación GenéticaRESUMEN
Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
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Biomarcadores de Tumor/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 21 , Subunidad beta Común de los Receptores de Citocinas/genética , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Leucemia Mieloide/genética , Reacción Leucemoide/genética , Mutación , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Síndrome de Down/diagnóstico , Factor de Transcripción GATA1/metabolismo , Regulación Leucémica de la Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/patología , Reacción Leucemoide/diagnóstico , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Fenotipo , Transcripción GenéticaRESUMEN
Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation.
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Conservación de la Sangre/métodos , Infecciones por Citomegalovirus/genética , Pruebas con Sangre Seca/métodos , Transcriptoma , Niño , Preescolar , Disfunción Cognitiva/diagnóstico , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/virología , Femenino , Humanos , Recién Nacido , Masculino , Enfermedad de la Neurona Motora/diagnóstico , Factores de Tiempo , Carga ViralRESUMEN
Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.
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Biomarcadores de Tumor/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Transcriptoma , Adulto , Biomarcadores de Tumor/metabolismo , Evolución Molecular , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Riesgo , Secuenciación Completa del Genoma/métodosRESUMEN
Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.
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Biomarcadores de Tumor/genética , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Persona de Mediana Edad , Adulto JovenRESUMEN
TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.
Asunto(s)
Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Técnicas de Cocultivo , Estudios de Cohortes , Análisis Mutacional de ADN , Resistencia a Antineoplásicos , Femenino , Expresión Génica , Estudios de Asociación Genética , Genómica , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Masculino , Ratones Endogámicos NOD , Ratones SCID , Mutación , Proteínas de Fusión Oncogénica/metabolismo , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Eliminación de Secuencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.
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ADN/genética , Exoma , Análisis de Secuencia de ADN/métodos , ADN/sangre , Biblioteca de Genes , Genómica/métodos , Técnicas de Genotipaje/métodos , Humanos , Tamaño de la MuestraRESUMEN
The DNA damage response (DDR) is a vast signaling network that is robustly activated by DNA double-strand breaks, the critical lesion induced by ionizing radiation (IR). Although much of this response operates at the protein level, a critical component of the network sustains many DDR branches by modulating the cellular transcriptome. Using deep sequencing, we delineated three layers in the transcriptional response to IR in human breast cancer cells: changes in the expression of genes encoding proteins or long noncoding RNAs, alterations in genomic binding by key transcription factors, and dynamics of epigenetic markers of active promoters and enhancers. We identified protein-coding and previously unidentified noncoding genes that were responsive to IR, and demonstrated that IR-induced transcriptional dynamics was mediated largely by the transcription factors p53 and nuclear factor κB (NF-κB) and was primarily dependent on the kinase ataxia-telangiectasia mutated (ATM). The resultant data set provides a rich resource for understanding a basic, underlying component of a critical cellular stress response.