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1.  A framework for modelling gene regulation which accommodates non-equilibrium mechanisms 
BMC Biology  2014;12(1):102.
Gene regulation has, for the most part, been quantitatively analysed by assuming that regulatory mechanisms operate at thermodynamic equilibrium. This formalism was originally developed to analyse the binding and unbinding of transcription factors from naked DNA in eubacteria. Although widely used, it has made it difficult to understand the role of energy-dissipating, epigenetic mechanisms, such as DNA methylation, nucleosome remodelling and post-translational modification of histones and co-regulators, which act together with transcription factors to regulate gene expression in eukaryotes.
Here, we introduce a graph-based framework that can accommodate non-equilibrium mechanisms. A gene-regulatory system is described as a graph, which specifies the DNA microstates (vertices), the transitions between microstates (edges) and the transition rates (edge labels). The graph yields a stochastic master equation for how microstate probabilities change over time. We show that this framework has broad scope by providing new insights into three very different ad hoc models, of steroid-hormone responsive genes, of inherently bounded chromatin domains and of the yeast PHO5 gene. We find, moreover, surprising complexity in the regulation of PHO5, which has not yet been experimentally explored, and we show that this complexity is an inherent feature of being away from equilibrium. At equilibrium, microstate probabilities do not depend on how a microstate is reached but, away from equilibrium, each path to a microstate can contribute to its steady-state probability. Systems that are far from equilibrium thereby become dependent on history and the resulting complexity is a fundamental challenge. To begin addressing this, we introduce a graph-based concept of independence, which can be applied to sub-systems that are far from equilibrium, and prove that history-dependent complexity can be circumvented when sub-systems operate independently.
As epigenomic data become increasingly available, we anticipate that gene function will come to be represented by graphs, as gene structure has been represented by sequences, and that the methods introduced here will provide a broader foundation for understanding how genes work.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0102-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4288563  PMID: 25475875
linear framework; Laplacian dynamics; thermodynamic formalism; non-equilibrium statistical mechanics; gene regulation; chromatin domains; steroid-hormone responsive genes; phosphate regulation; PHO5; product graph; independence
2.  Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen 
PLoS Computational Biology  2014;10(9):e1003814.
Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest.
Author Summary
Mathematical models which aim to describe cellular signaling start from constructing an interaction network of effectors, mediators and their effected target proteins. Several developments came up making it easier to put these links together. Besides tediously assembling knowledge from textbooks and research articles, experimental high-throughput methods were established like Yeast-2-Hybrid assays or Fluorescence Emission Resonance Transfer. However, these methods do not elucidate the effect of such interactions. We aimed inferring if an interaction in a specific cellular context is rather activating or inhibiting. We used cellular phenotypes of a genome-wide RNAi knockdown screen of live cells to identify such activating and inhibiting effects of protein interactions. The rationale behind it is that activating protein interactions should lead to similar phenotypes when their respective genes are knocked down, whereas an inhibiting protein interaction should lead to dissimilar phenotypes. Exemplarily, we applied our method to a phenotype screen of perturbed HeLa cells. Our predictions effectively reproduced textbook relationships between proteins or domains when comparing the predicted effects with pairs of effectors, receptors, kinases, phosphatases and of general signalling modules. The presented computational approach is generic and may enable elucidating the effects of studied interactions also of other cellular systems under more specific conditions.
PMCID: PMC4178005  PMID: 25255318
3.  Data-Derived Modeling Characterizes Plasticity of MAPK Signaling in Melanoma 
PLoS Computational Biology  2014;10(9):e1003795.
The majority of melanomas have been shown to harbor somatic mutations in the RAS-RAF-MEK-MAPK and PI3K-AKT pathways, which play a major role in regulation of proliferation and survival. The prevalence of these mutations makes these kinase signal transduction pathways an attractive target for cancer therapy. However, tumors have generally shown adaptive resistance to treatment. This adaptation is achieved in melanoma through its ability to undergo neovascularization, migration and rearrangement of signaling pathways. To understand the dynamic, nonlinear behavior of signaling pathways in cancer, several computational modeling approaches have been suggested. Most of those models require that the pathway topology remains constant over the entire observation period. However, changes in topology might underlie adaptive behavior to drug treatment. To study signaling rearrangements, here we present a new approach based on Fuzzy Logic (FL) that predicts changes in network architecture over time. This adaptive modeling approach was used to investigate pathway dynamics in a newly acquired experimental dataset describing total and phosphorylated protein signaling over four days in A375 melanoma cell line exposed to different kinase inhibitors. First, a generalized strategy was established to implement a parameter-reduced FL model encoding non-linear activity of a signaling network in response to perturbation. Next, a literature-based topology was generated and parameters of the FL model were derived from the full experimental dataset. Subsequently, the temporal evolution of model performance was evaluated by leaving time-defined data points out of training. Emerging discrepancies between model predictions and experimental data at specific time points allowed the characterization of potential network rearrangement. We demonstrate that this adaptive FL modeling approach helps to enhance our mechanistic understanding of the molecular plasticity of melanoma.
Author Summary
Signal transduction pathways can be described as static routes, transmitting extrinsic signals to the nucleus to induce a transcriptional response. In contrast to this reductionist view, the emerging paradigm is that signaling networks undergo dynamic crosstalk, both in disease and physiological conditions. To understand complex pathway behavior, it is necessary to develop methods to identify pathway interactions that are active as a consequence of stimuli and, importantly, to describe their evolution in time. To that end, we developed a method relying on prior knowledge networks in order to predict signaling crosstalk evolution, in response to perturbation and over time. The challenge we addressed was to establish a method dependent on information related to the topology of reported interactions, and not their mechanistic characteristics, and at the same time complex enough to reproduce the behavior of the signaling intermediates. The work presented here demonstrates that such an approach can be used to predict mechanisms that melanoma uses to rearrange its signaling and maintain its abnormal proliferation upon treatment.
PMCID: PMC4154640  PMID: 25188314
4.  Estimating the activity of transcription factors by the effect on their target genes 
Bioinformatics  2014;30(17):i401-i407.
Motivation: Understanding regulation of transcription is central for elucidating cellular regulation. Several statistical and mechanistic models have come up the last couple of years explaining gene transcription levels using information of potential transcriptional regulators as transcription factors (TFs) and information from epigenetic modifications. The activity of TFs is often inferred by their transcription levels, promoter binding and epigenetic effects. However, in principle, these methods do not take hard-to-measure influences such as post-transcriptional modifications into account.
Results: For TFs, we present a novel concept circumventing this problem. We estimate the regulatory activity of TFs using their cumulative effects on their target genes. We established our model using expression data of 59 cell lines from the National Cancer Institute. The trained model was applied to an independent expression dataset of melanoma cells yielding excellent expression predictions and elucidated regulation of melanogenesis.
Availability and implementation: Using mixed-integer linear programming, we implemented a switch-like optimization enabling a constrained but optimal selection of TFs and optimal model selection estimating their effects. The method is generic and can also be applied to further regulators of transcription.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC4147899  PMID: 25161226
5.  Network topology-based detection of differential gene regulation and regulatory switches in cell metabolism and signaling 
BMC Systems Biology  2014;8:56.
Common approaches to pathway analysis treat pathways merely as lists of genes disregarding their topological structures, that is, ignoring the genes' interactions on which a pathway's cellular function depends. In contrast, PathWave has been developed for the analysis of high-throughput gene expression data that explicitly takes the topology of networks into account to identify both global dysregulation of and localized (switch-like) regulatory shifts within metabolic and signaling pathways. For this purpose, it applies adjusted wavelet transforms on optimized 2D grid representations of curated pathway maps.
Here, we present the new version of PathWave with several substantial improvements including a new method for optimally mapping pathway networks unto compact 2D lattice grids, a more flexible and user-friendly interface, and pre-arranged 2D grid representations. These pathway representations are assembled for several species now comprising H. sapiens, M. musculus, D. melanogaster, D. rerio, C. elegans, and E. coli. We show that PathWave is more sensitive than common approaches and apply it to RNA-seq expression data, identifying crucial metabolic pathways in lung adenocarcinoma, as well as microarray expression data, identifying pathways involved in longevity of Drosophila.
PathWave is a generic method for pathway analysis complementing established tools like GSEA, and the update comprises efficient new features. In contrast to the tested commonly applied approaches which do not take network topology into account, PathWave enables identifying pathways that are either known be involved in or very likely associated with such diverse conditions as human lung cancer or aging of D. melanogaster. The PathWave R package is freely available at
PMCID: PMC4031158  PMID: 24886210
Pathway analysis; Network topology; Pathway networks; Gene expression
6.  Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma 
Jones, David T.W. | Hutter, Barbara | Jäger, Natalie | Korshunov, Andrey | Kool, Marcel | Warnatz, Hans-Jörg | Zichner, Thomas | Lambert, Sally R. | Ryzhova, Marina | Quang, Dong Anh Khuong | Fontebasso, Adam M. | Stütz, Adrian M. | Hutter, Sonja | Zuckermann, Marc | Sturm, Dominik | Gronych, Jan | Lasitschka, Bärbel | Schmidt, Sabine | Şeker-Cin, Huriye | Witt, Hendrik | Sultan, Marc | Ralser, Meryem | Northcott, Paul A. | Hovestadt, Volker | Bender, Sebastian | Pfaff, Elke | Stark, Sebastian | Faury, Damien | Schwartzentruber, Jeremy | Majewski, Jacek | Weber, Ursula D. | Zapatka, Marc | Raeder, Benjamin | Schlesner, Matthias | Worth, Catherine L. | Bartholomae, Cynthia C. | von Kalle, Christof | Imbusch, Charles D. | Radomski, Sylwester | Lawerenz, Chris | van Sluis, Peter | Koster, Jan | Volckmann, Richard | Versteeg, Rogier | Lehrach, Hans | Monoranu, Camelia | Winkler, Beate | Unterberg, Andreas | Herold-Mende, Christel | Milde, Till | Kulozik, Andreas E. | Ebinger, Martin | Schuhmann, Martin U. | Cho, Yoon-Jae | Pomeroy, Scott L. | von Deimling, Andreas | Witt, Olaf | Taylor, Michael D. | Wolf, Stephan | Karajannis, Matthias A. | Eberhart, Charles G. | Scheurlen, Wolfram | Hasselblatt, Martin | Ligon, Keith L. | Kieran, Mark W. | Korbel, Jan O. | Yaspo, Marie-Laure | Brors, Benedikt | Felsberg, Jörg | Reifenberger, Guido | Collins, V. Peter | Jabado, Nada | Eils, Roland | Lichter, Peter | Pfister, Stefan M.
Nature genetics  2013;45(8):927-932.
Pilocytic astrocytoma, the most common childhood brain tumor1, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations2. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression3 and often becoming a chronic disease with substantial morbidities4.
Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n=73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and novel NTRK2 fusion genes in non-cerebellar tumors. New BRAF activating changes were also observed. MAPK pathway alterations affected 100% of tumors analyzed, with no other significant mutations, indicating pilocytic astrocytoma as predominantly a single-pathway disease.
Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in NF15. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.
PMCID: PMC3951336  PMID: 23817572
7.  Signatures of mutational processes in human cancer 
Alexandrov, Ludmil B. | Nik-Zainal, Serena | Wedge, David C. | Aparicio, Samuel A.J.R. | Behjati, Sam | Biankin, Andrew V. | Bignell, Graham R. | Bolli, Niccolo | Borg, Ake | Børresen-Dale, Anne-Lise | Boyault, Sandrine | Burkhardt, Birgit | Butler, Adam P. | Caldas, Carlos | Davies, Helen R. | Desmedt, Christine | Eils, Roland | Eyfjörd, Jórunn Erla | Foekens, John A. | Greaves, Mel | Hosoda, Fumie | Hutter, Barbara | Ilicic, Tomislav | Imbeaud, Sandrine | Imielinsk, Marcin | Jäger, Natalie | Jones, David T.W. | Jones, David | Knappskog, Stian | Kool, Marcel | Lakhani, Sunil R. | López-Otín, Carlos | Martin, Sancha | Munshi, Nikhil C. | Nakamura, Hiromi | Northcott, Paul A. | Pajic, Marina | Papaemmanuil, Elli | Paradiso, Angelo | Pearson, John V. | Puente, Xose S. | Raine, Keiran | Ramakrishna, Manasa | Richardson, Andrea L. | Richter, Julia | Rosenstiel, Philip | Schlesner, Matthias | Schumacher, Ton N. | Span, Paul N. | Teague, Jon W. | Totoki, Yasushi | Tutt, Andrew N.J. | Valdés-Mas, Rafael | van Buuren, Marit M. | van ’t Veer, Laura | Vincent-Salomon, Anne | Waddell, Nicola | Yates, Lucy R. | Zucman-Rossi, Jessica | Futreal, P. Andrew | McDermott, Ultan | Lichter, Peter | Meyerson, Matthew | Grimmond, Sean M. | Siebert, Reiner | Campo, Elías | Shibata, Tatsuhiro | Pfister, Stefan M. | Campbell, Peter J. | Stratton, Michael R.
Nature  2013;500(7463):415-421.
All cancers are caused by somatic mutations. However, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here, we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, kataegis, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer with potential implications for understanding of cancer etiology, prevention and therapy.
PMCID: PMC3776390  PMID: 23945592
8.  Decision-Tree Based Model Analysis for Efficient Identification of Parameter Relations Leading to Different Signaling States 
PLoS ONE  2013;8(12):e82593.
In systems biology, a mathematical description of signal transduction processes is used to gain a more detailed mechanistic understanding of cellular signaling networks. Such models typically depend on a number of parameters that have different influence on the model behavior. Local sensitivity analysis is able to identify parameters that have the largest effect on signaling strength. Bifurcation analysis shows on which parameters a qualitative model response depends. Most methods for model analysis are intrinsically univariate. They typically cannot consider combinations of parameters since the search space for such analysis would be too large. This limitation is important since activation of a signaling pathway often relies on multiple rather than on single factors. Here, we present a novel method for model analysis that overcomes this limitation. As input to a model defined by a system of ordinary differential equations, we consider parameters for initial chemical species concentrations. The model is used to simulate the system response, which is then classified into pre-defined classes (e.g., active or not active). This is combined with a scan of the parameter space. Parameter sets leading to a certain system response are subjected to a decision tree algorithm, which learns conditions that lead to this response. We compare our method to two alternative multivariate approaches to model analysis: analytical solution for steady states combined with a parameter scan, and direct Lyapunov exponent (DLE) analysis. We use three previously published models including a model for EGF receptor internalization and two apoptosis models to demonstrate the power of our approach. Our method reproduces critical parameter relations previously obtained by both steady-state and DLE analysis while being more generally applicable and substantially less computationally expensive. The method can be used as a general tool to predict multivariate control strategies for pathway activation and to suggest strategies for drug intervention.
PMCID: PMC3867358  PMID: 24367526
9.  Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas 
Genome Biology  2013;14(12):r137.
High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear.
We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability.
Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas.
PMCID: PMC4054884  PMID: 24345474
10.  Genome sequencing: a systematic review of health economic evidence 
Recently the sequencing of the human genome has become a major biological and clinical research field. However, the public health impact of this new technology with focus on the financial effect is not yet to be foreseen. To provide an overview of the current health economic evidence for genome sequencing, we conducted a thorough systematic review of the literature from 17 databases. In addition, we conducted a hand search. Starting with 5 520 records we ultimately included five full-text publications and one internet source, all focused on cost calculations. The results were very heterogeneous and, therefore, difficult to compare. Furthermore, because the methodology of the publications was quite poor, the reliability and validity of the results were questionable. The real costs for the whole sequencing workflow, including data management and analysis, remain unknown. Overall, our review indicates that the current health economic evidence for genome sequencing is quite poor. Therefore, we listed aspects that needed to be considered when conducting health economic analyses of genome sequencing. Thereby, specifics regarding the overall aim, technology, population, indication, comparator, alternatives after sequencing, outcomes, probabilities, and costs with respect to genome sequencing are discussed. For further research, at the outset, a comprehensive cost calculation of genome sequencing is needed, because all further health economic studies rely on valid cost data. The results will serve as an input parameter for budget-impact analyses or cost-effectiveness analyses.
PMCID: PMC3874598  PMID: 24330507
Genome; Sequencing; Health economics; Cost analysis
11.  Hypermutation of the Inactive X Chromosome Is a Frequent Event in Cancer 
Cell  2013;155(3):567-581.
Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.
Graphical Abstract
•X chromosome has up to 4× more mutations than the autosomes in female cancer genomes•Hypermutations only affect the inactive X chromosome•X hypermutation involves somatic point mutations and indels, but not germline mutations•No X hypermutation is found in clonal expansions of normal or premalignant cells
A comparison of 402 cancer genomes identifies a surprisingly high level of somatic mutations in the inactive X chromosome of female cancer genomes. As hypermutability of the inactive X was not observed in clonal hematopoietic progenitor or preleukemic samples, it is likely that it may be a contributing factor to tumorigenesis.
PMCID: PMC3898475  PMID: 24139898
12.  Live cell assays to identify regulators of ER to Golgi trafficking 
Traffic (Copenhagen, Denmark)  2012;13(3):416-432.
We applied fluorescence microscopy based quantitative assays to living cells to identify regulators of ER to Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors, which influence Golgi to ER re-localization of GalT-CFP after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when over-expressed for a role in ER to Golgi trafficking. Nine of them interfered with the rate of BFA induced redistribution of GalT-CFP from the Golgi complex to the ER, 6 of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e., Golgi complex) after BFA wash-out, and 6 of them were positive effectors in both assays. Notably, our live cell approach captures regulator function in ER to Golgi trafficking, that were missed in previous fixed cell assays; as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.
PMCID: PMC3711101  PMID: 22132776
Brefeldin A (BFA); GalT; ER to Golgi trafficking; YIPF; GOT1B; USE1; SACM1L
13.  Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies 
PLoS ONE  2013;8(6):e66621.
The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes.
PMCID: PMC3679043  PMID: 23776689
14.  ICGC PedBrain: Dissecting the genomic complexity underlying medulloblastoma 
Jones, David TW | Jäger, Natalie | Kool, Marcel | Zichner, Thomas | Hutter, Barbara | Sultan, Marc | Cho, Yoon-Jae | Pugh, Trevor J | Hovestadt, Volker | Stütz, Adrian M | Rausch, Tobias | Warnatz, Hans-Jörg | Ryzhova, Marina | Bender, Sebastian | Sturm, Dominik | Pleier, Sabrina | Cin, Huriye | Pfaff, Elke | Sieber, Laura | Wittmann, Andrea | Remke, Marc | Witt, Hendrik | Hutter, Sonja | Tzaridis, Theophilos | Weischenfeldt, Joachim | Raeder, Benjamin | Avci, Meryem | Amstislavskiy, Vyacheslav | Zapatka, Marc | Weber, Ursula D | Wang, Qi | Lasitschka, Bärbel | Bartholomae, Cynthia C | Schmidt, Manfred | von Kalle, Christof | Ast, Volker | Lawerenz, Chris | Eils, Jürgen | Kabbe, Rolf | Benes, Vladimir | van Sluis, Peter | Koster, Jan | Volckmann, Richard | Shih, David | Betts, Matthew J | Russell, Robert B | Coco, Simona | Tonini, Gian Paolo | Schüller, Ulrich | Hans, Volkmar | Graf, Norbert | Kim, Yoo-Jin | Monoranu, Camelia | Roggendorf, Wolfgang | Unterberg, Andreas | Herold-Mende, Christel | Milde, Till | Kulozik, Andreas E | von Deimling, Andreas | Witt, Olaf | Maass, Eberhard | Rössler, Jochen | Ebinger, Martin | Schuhmann, Martin U | Frühwald, Michael C | Hasselblatt, Martin | Jabado, Nada | Rutkowski, Stefan | von Bueren, André O | Williamson, Dan | Clifford, Steven C | McCabe, Martin G | Collins, V. Peter | Wolf, Stephan | Wiemann, Stefan | Lehrach, Hans | Brors, Benedikt | Scheurlen, Wolfram | Felsberg, Jörg | Reifenberger, Guido | Northcott, Paul A | Taylor, Michael D | Meyerson, Matthew | Pomeroy, Scott L | Yaspo, Marie-Laure | Korbel, Jan O | Korshunov, Andrey | Eils, Roland | Pfister, Stefan M | Lichter, Peter
Nature  2012;488(7409):100-105.
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 displays tremendous biological and clinical heterogeneity1. 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 discriminated2,3. WNT tumours, displaying activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens4. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis2. Group 3 & 4 tumours are molecularly less well-characterised, and also present the greatest clinical challenges2,3,5. 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. Tetraploidy was identified as a frequent early event in Group 3 & 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 the first medulloblastoma fusion genes. 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 & 4 patients.
PMCID: PMC3662966  PMID: 22832583
15.  Genome Sequencing of Pediatric Medulloblastoma Links Catastrophic DNA Rearrangements with TP53 Mutations 
Cell  2012;148(1-2):59-71.
Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.
PMCID: PMC3332216  PMID: 22265402
16.  miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus 
PLoS ONE  2012;7(12):e52555.
miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.
PMCID: PMC3527550  PMID: 23285084
17.  Time-Lapse Imaging of Neuroblastoma Cells to Determine Cell Fate upon Gene Knockdown 
PLoS ONE  2012;7(12):e50988.
Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at
PMCID: PMC3521006  PMID: 23251412
19.  Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment 
Cell host & microbe  2011;9(1):32-45.
Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.
PMCID: PMC3433060  PMID: 21238945
20.  Nonrigid Registration of 2-D and 3-D Dynamic Cell Nuclei Images for Improved Classification of Subcellular Particle Motion 
The observed motion of subcellular particles in fluorescence microscopy image sequences of live cells is generally a superposition of the motion and deformation of the cell and the motion of the particles. Decoupling the two types of movements to enable accurate classification of the particle motion requires the application of registration algorithms. We have developed an intensity-based approach for nonrigid registration of multi-channel microscopy image sequences of cell nuclei. First, based on 3-D synthetic images we demonstrate that cell nucleus deformations change the observed motion types of particles and that our approach allows to recover the original motion. Second, we have successfully applied our approach to register 2-D and 3-D real microscopy image sequences. A quantitative experimental comparison with previous approaches for nonrigid registration of cell microscopy has also been performed.
PMCID: PMC3282047  PMID: 20840894
Biomedical image processing; image sequence analysis; microscopy; registration
21.  Multi-Parametric Analysis and Modeling of Relationships between Mitochondrial Morphology and Apoptosis 
PLoS ONE  2012;7(1):e28694.
Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis.
PMCID: PMC3260148  PMID: 22272225
22.  Normalizing for individual cell population context in the analysis of high-content cellular screens 
BMC Bioinformatics  2011;12:485.
High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.
We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.
Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.
PMCID: PMC3259109  PMID: 22185194
23.  Autoantibodies against the exocrine pancreas in autoimmune pancreatitis: gene and protein expression profiling and immunoassays identify pancreatic enzymes as a major target of the inflammatory process 
Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas.
In order to explore key targets of the inflammatory process we analysed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, Western blot and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared to those of 8 patients with non-AIP chronic pancreatitis (CP).
Expression profiling revealed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry demonstrated a near-loss of trypsin positive acinar cells, which was also confirmed by Western blotting. The serum of AIP patients contained high titres of autoantibodies against the trypsinogens PRSS1, and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals we found similar protein patterns and a reduction in trypsinogen.
These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.
PMCID: PMC3099227  PMID: 20407433
autoimmune pancreatitis; chronic pancreatitis; trypsinogen; proteomics; transcriptomics; autoantibody
24.  Enhancers regulate progression of development in mammalian cells 
Nucleic Acids Research  2011;39(20):8689-8702.
During development and differentiation of an organism, accurate gene regulation is central for cells to maintain and balance their differentiation processes. Transcriptional interactions between cis-acting DNA elements such as promoters and enhancers are the basis for precise and balanced transcriptional regulation. We identified modules of combinations of binding sites in proximal and distal regulatory regions upstream of all transcription start sites (TSSs) in silico and applied these modules to gene expression time-series of mouse embryonic development and differentiation of human stem cells. In addition to tissue-specific regulation controlled by combinations of transcription factors (TFs) binding at promoters, we observed that in particular the combination of TFs binding at promoters together with TFs binding at the respective enhancers regulate highly specifically temporal progression during development: whereas 40% of TFs were specific for time intervals, 79% of TF pairs and even 97% of promoter–enhancer modules showed specificity for single time intervals of the human stem cells. Predominantly SP1 and E2F contributed to temporal specificity at promoters and the forkhead (FOX) family of TFs at enhancer regions. Altogether, we characterized three classes of TFs: with binding sites being enriched at the TSS (like SP1), depleted at the TSS (like FOX), and rather uniformly distributed.
PMCID: PMC3203619  PMID: 21785139
25.  Formulating multicellular models of metabolism in tissues: application to energy metabolism in the human brain 
Nature biotechnology  2010;28(12):1279-1285.
A workflow is presented that integrates gene expression data, proteomic data, and literature-based manual curation to construct multicellular, tissue-specific models of human brain energy metabolism that recapitulate metabolic interactions between astrocytes and various neuron types. Three analyses are applied for gene identification, analysis of omics data, and analysis of physiological states. First, we identify glutamate decarboxylase as a target that may contribute to cell-type and regional specificity in Alzheimer’s disease. Second, the decreased metabolic rate seen in affected brain regions in Alzheimer’s disease is consistent with a suppression of central metabolic gene expression in histopathologically normal neurons. Third, we identify pathways in cholinergic neurons that couple mitochondrial metabolism and cytosolic acetylcholine production, and subsequently find that cholinergic neurotransmission accounts for ∼3% of brain neurotransmission. Constraint-based modeling can thus contribute to the study and analysis of multicellular metabolic processes in human tissues, and provide detailed mechanistic insight into high-throughput data analysis.
PMCID: PMC3140076  PMID: 21102456

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