1,721,108 research outputs found
Mutations driving CLL and their evolution in progression and relapse
No full textWhich genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized putative cancer drivers (RPS15, IKZF3), and collectively identify RNA processing and export, MYC activity, and MAPK signalling as central pathways involved in CLL. Clonality analysis of this large data set further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing data sets of clinically informative samples enable the discovery of novel genes associated with cancer, the network of relationships between the driver events, and their impact on disease relapse and clinical outcome.National Human Genome Research Institute (U.S.) (Grant U54HG003067
Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction
No full textMyocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, the role of inheritance is substantially greater. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families whereas common variants at more than 45 loci have been associated with MI risk in the population. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C3. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.National Heart, Lung, and Blood Institute (Grants RC2 HL-103010, RC2 HL-102923, RC2 HL-102924, RC2 HL-102925, and RC2 HL-102926)National Human Genome Research Institute (U.S.) (Grant 5U54HG003067-1)National Institutes of Health (U.S.) (Grants P01 HL076491 and P01 HL098055
Brave New Genome
No full textFifty years ago, microbiologists sparked the recombinant-DNA revolution with the discovery that bacteria have innate immune systems based on restriction enzymes. These enzymes bind and cut invading viral genomes at specific short sequences, and scientists rapidly repurposed them to cut and paste DNA in vitro — transforming biologic science and giving rise to the biotechnology industry
Genomic Characterization of Brain Metastases Reveals Branched Evolution and Potential Therapeutic Targets
No full textBrain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed wholeexome sequencing of 86 matched brain metastases, primary tumors and normal tissue. In all
clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched
primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases.Massachusetts Institute of Technology. Department of BiologyNational Institutes of Health (U.S.) (National Human Genome Research Institutes of Health Large-scale Sequencing and Analysis Center. Grant U54 HG003067
Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition
No full textResistance to the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.University of Texas M.D. Anderson Cancer Center (Support Grant CA016672)National Science Foundation (U.S.) (DMR-1310266)Harvard University. Materials Research Science and Engineering Center (DMR-1420570)National Natural Science Foundation (China) (81372496
Single Guide RNA Library Design and Construction
No full textThis protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice
Viral Packaging and Cell Culture for CRISPR-Based Screens
No full textThis protocol describes how to perform the tissue culture and high-throughput sequencing library preparation for a CRISPR-based screen. First, pantropic lentivirus is prepared from a sgRNA plasmid pool and applied to the target cells. Following antibiotic selection and a harvest of the initial population, cells are then cultured under the desired screening condition(s) for 14 population doublings. sgRNA barcode sequences integrated in the genomic DNA of each cell population are amplified and subject to high-throughput sequencing. Guidelines for downstream analysis of the sequencing data are also provided
Large-Scale Single Guide RNA Library Construction and Use for CRISPR–Cas9-Based Genetic Screens
No full textThe ability to systematically disrupt genes serves as a powerful tool for understanding their function. The programmable CRISPR–Cas9 system enables efficient targeting of large numbers of genes through the use of single guide RNA (sgRNA) libraries. In cultured mammalian cells, collections of knockout mutants can be readily generated by means of transduction of Cas9–sgRNA lentiviral pools, screened for a phenotype of interest, and counted using high-throughput DNA sequencing. This technique represents the first general method for undertaking systematic loss-of-function genetic screens in mammalian cells. Here, we introduce the methodology and rationale for conducting CRISPR-based screens, focusing on distinguishing positive and negative selection strategies.National Institutes of Health (U.S.) (Grant CA103866)National Human Genome Research Institute (U.S.) (Grant 2U54HG003067-10)Broad Institute of MIT and HarvardNational Science Foundation (U.S.
Positional specificity of different transcription factor classes within enhancers
Full text linkGene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers. Keywords: transcription factor binding; gene regulation; genomics; chromatin structureNational Human Genome Research Institute (U.S.) (Grant 2U54HG003067-10)National Institute of General Medical Sciences (U.S.) (Grant T32GM007753
Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments
No full textHi-C experiments explore the 3D structure of the genome, generating terabases of data to create high-resolution contact maps. Here, we introduce Juicer, an open-source tool for analyzing terabase-scale Hi-C datasets. Juicer allows users without a computational background to transform raw sequence data into normalized contact maps with one click. Juicer produces a hic file containing compressed contact matrices at many resolutions, facilitating visualization and analysis at multiple scales. Structural features, such as loops and domains, are automatically annotated. Juicer is available as open source software at http://aidenlab.org/juicer/.National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008540)National Science Foundation (U.S.) Physics Frontier Center (Grant PHY-1427654)National Human Genome Research Institute (U.S.) (Grant HG006193)Robert A. Welch Foundation (Grant Q-1866)Cancer Prevention and Research Institute of Texas (Scholar Award R1304)NVIDIA Corporation (Research Center Award)IBM (University Challenge Award)Google (Research Award)Baylor College of Medicine (McNair Medical Institute Scholar Award)National Human Genome Research Institute (U.S.) (Grant HG003067)National Institutes of Health (U.S.) (4D Nucleome Grant U01HL130010
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