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1.  Comparative Analysis and Modeling of the Severity of Steatohepatitis in DDC-Treated Mouse Strains 
PLoS ONE  2014;9(10):e111006.
Background
Non-alcoholic fatty liver disease (NAFLD) has a broad spectrum of disease states ranging from mild steatosis characterized by an abnormal retention of lipids within liver cells to steatohepatitis (NASH) showing fat accumulation, inflammation, ballooning and degradation of hepatocytes, and fibrosis. Ultimately, steatohepatitis can result in liver cirrhosis and hepatocellular carcinoma.
Methodology and Results
In this study we have analyzed three different mouse strains, A/J, C57BL/6J, and PWD/PhJ, that show different degrees of steatohepatitis when administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) containing diet. RNA-Seq gene expression analysis, protein analysis and metabolic profiling were applied to identify differentially expressed genes/proteins and perturbed metabolite levels of mouse liver samples upon DDC-treatment. Pathway analysis revealed alteration of arachidonic acid (AA) and S-adenosylmethionine (SAMe) metabolism upon other pathways. To understand metabolic changes of arachidonic acid metabolism in the light of disease expression profiles a kinetic model of this pathway was developed and optimized according to metabolite levels. Subsequently, the model was used to study in silico effects of potential drug targets for steatohepatitis.
Conclusions
We identified AA/eicosanoid metabolism as highly perturbed in DDC-induced mice using a combination of an experimental and in silico approach. Our analysis of the AA/eicosanoid metabolic pathway suggests that 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin D2 (PGD2) are perturbed in DDC mice. We further demonstrate that a dynamic model can be used for qualitative prediction of metabolic changes based on transcriptomics data in a disease-related context. Furthermore, SAMe metabolism was identified as being perturbed due to DDC treatment. Several genes as well as some metabolites of this module show differences between A/J and C57BL/6J on the one hand and PWD/PhJ on the other.
doi:10.1371/journal.pone.0111006
PMCID: PMC4210132  PMID: 25347188
2.  Influence of RNA extraction methods and library selection schemes on RNA-seq data 
BMC Genomics  2014;15(1):675.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-675) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-675
PMCID: PMC4148917  PMID: 25113896
RNA-Seq; RNA extraction; rRNA depletion; poly(A)+ selection; Intronic reads
3.  ARH-seq: identification of differential splicing in RNA-seq data 
Nucleic Acids Research  2014;42(14):e110.
The computational prediction of alternative splicing from high-throughput sequencing data is inherently difficult and necessitates robust statistical measures because the differential splicing signal is overlaid by influencing factors such as gene expression differences and simultaneous expression of multiple isoforms amongst others. In this work we describe ARH-seq, a discovery tool for differential splicing in case–control studies that is based on the information-theoretic concept of entropy. ARH-seq works on high-throughput sequencing data and is an extension of the ARH method that was originally developed for exon microarrays. We show that the method has inherent features, such as independence of transcript exon number and independence of differential expression, what makes it particularly suited for detecting alternative splicing events from sequencing data. In order to test and validate our workflow we challenged it with publicly available sequencing data derived from human tissues and conducted a comparison with eight alternative computational methods. In order to judge the performance of the different methods we constructed a benchmark data set of true positive splicing events across different tissues agglomerated from public databases and show that ARH-seq is an accurate, computationally fast and high-performing method for detecting differential splicing events.
doi:10.1093/nar/gku495
PMCID: PMC4132698  PMID: 24920826
4.  The Nerve Growth Factor Receptor CD271 Is Crucial to Maintain Tumorigenicity and Stem-Like Properties of Melanoma Cells 
PLoS ONE  2014;9(5):e92596.
Background
Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133.
Results
Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271+ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133+ sub-set that in addition comprised a fraction of cells of a CD271+/CD133+ phenotype. Those cells were found in the label-retaining fraction and in vitro deduced from CD271+ but not CD271 knock-down cells.
Conclusions
Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.
doi:10.1371/journal.pone.0092596
PMCID: PMC4010406  PMID: 24799129
5.  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.
doi:10.1038/ng.2682
PMCID: PMC3951336  PMID: 23817572
6.  Hypermutation of the Inactive X Chromosome Is a Frequent Event in Cancer 
Cell  2013;155(3):567-581.
Summary
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
Highlights
•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.
doi:10.1016/j.cell.2013.09.042
PMCID: PMC3898475  PMID: 24139898
7.  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.
Summary
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.
doi:10.1038/nature11284
PMCID: PMC3662966  PMID: 22832583
9.  Matrin 3 Binds and Stabilizes mRNA 
PLoS ONE  2011;6(8):e23882.
Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.
doi:10.1371/journal.pone.0023882
PMCID: PMC3157474  PMID: 21858232
10.  A High-Resolution Anatomical Atlas of the Transcriptome in the Mouse Embryo 
PLoS Biology  2011;9(1):e1000582.
The manuscript describes the “digital transcriptome atlas” of the developing mouse embryo, a powerful resource to determine co-expression of genes, to identify cell populations and lineages and to identify functional associations between genes relevant to development and disease.
Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.
Author Summary
In situ hybridization (ISH) can be used to visualize gene expression in cells and tissues in their native context. High-throughput ISH using nonradioactive RNA probes allowed the Eurexpress consortium to generate a comprehensive, interactive, and freely accessible digital gene expression atlas, the Eurexpress transcriptome atlas (http://www.eurexpress.org), of the E14.5 mouse embryo. Expression data for over 15,000 genes were annotated for hundreds of anatomical structures, thus allowing us to systematically identify tissue-specific and tissue-overlapping gene networks. We illustrate the value of the Eurexpress atlas by finding novel regional subdivisions in the developing brain. We also use the transcriptome atlas to allocate specific components of the complex Wnt signaling pathway to kidney development, and we identify regionally expressed genes in liver that may be markers of hematopoietic stem cell differentiation.
doi:10.1371/journal.pbio.1000582
PMCID: PMC3022534  PMID: 21267068
11.  Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters 
Nucleic Acids Research  2010;38(18):6112-6123.
Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation.
doi:10.1093/nar/gkq402
PMCID: PMC2952857  PMID: 20494980
12.  Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments 
Nucleic Acids Research  2010;38(10):e112.
Alternative splicing, polyadenylation of pre-messenger RNA molecules and differential promoter usage can produce a variety of transcript isoforms whose respective expression levels are regulated in time and space, thus contributing specific biological functions. However, the repertoire of mammalian alternative transcripts and their regulation are still poorly understood. Second-generation sequencing is now opening unprecedented routes to address the analysis of entire transcriptomes. Here, we developed methods that allow the prediction and quantification of alternative isoforms derived solely from exon expression levels in RNA-Seq data. These are based on an explicit statistical model and enable the prediction of alternative isoforms within or between conditions using any known gene annotation, as well as the relative quantification of known transcript structures. Applying these methods to a human RNA-Seq dataset, we validated a significant fraction of the predictions by RT-PCR. Data further showed that these predictions correlated well with information originating from junction reads. A direct comparison with exon arrays indicated improved performances of RNA-Seq over microarrays in the prediction of skipped exons. Altogether, the set of methods presented here comprehensively addresses multiple aspects of alternative isoform analysis. The software is available as an open-source R-package called Solas at http://cmb.molgen.mpg.de/2ndGenerationSequencing/Solas/.
doi:10.1093/nar/gkq041
PMCID: PMC2879520  PMID: 20150413
13.  Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules 
Molecular Biology of the Cell  2007;18(4):1385-1396.
Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.
doi:10.1091/mbc.E06-12-1120
PMCID: PMC1838996  PMID: 17392519
14.  Gene expression variation in Down's syndrome mice allows prioritization of candidate genes 
Genome Biology  2007;8(5):R91.
RNA from eight Ts65Dn mice (a model of Down syndrome) and eight euploid mice were analysed by real-time PCR to examine inter-individual gene expression levels as a function of trisomy.
Background
Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction.
Results
In pooled RNAs, we confirmed that trisomic/euploid gene expression ratios were close to 1.5. However, we observed that inter-individual gene expression levels spanned a broad range of values. We identified three categories of genes: genes with expression levels consistently higher in Ts65Dn than in euploids (9, 17, and 7 genes in cerebellum, cortex, and midbrain, respectively); genes whose expression levels partially overlap between the two groups (10, 9, and 14 genes); and genes with intermingled expression, which cannot be used to differentiate trisomics from euploids (12, 5 and 9 genes). Of the genes in the first category, App, Cbr1, and Mrps6 exhibited tight regulation in the three tissues and are therefore attractive candidates for further research.
Conclusion
This is the first analysis addressing inter-individual gene expression levels as a function of trisomy. We propose a strategy allowing discrimination between candidates for the constant features of DS and those genes that may contribute to the partially penetrant signs of DS.
doi:10.1186/gb-2007-8-5-r91
PMCID: PMC1929163  PMID: 17531092
15.  Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins 
BMC Genomics  2006;7:155.
Background
Trisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins.
Results
We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized.
Conclusion
The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.
doi:10.1186/1471-2164-7-155
PMCID: PMC1526728  PMID: 16780588

Results 1-16 (16)