Publications by authors named "Marc Sultan"

29 Publications

  • Page 1 of 1

Detection of CSF1 rearrangements deleting the 3' UTR in tenosynovial giant cell tumors.

Genes Chromosomes Cancer 2020 02 12;59(2):96-105. Epub 2019 Sep 12.

Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.

Tenosynovial giant cell tumors (TGCTs) are characterized by rearrangements of CSF1, thought to drive overexpression of macrophage colony-stimulating factor (CSF1), thereby promoting tumor growth and recruitment of non-neoplastic mononuclear and multinucleated inflammatory cells. While fusions to collagen promoters have been described, the mechanism of CSF1 overexpression has been unclear in a majority of cases. Two cohorts of TGCT were investigated for CSF1 rearrangements using fluorescence in situ hybridization (FISH) and either RNA-seq or DNA-seq with Sanger validation. The study comprised 39 patients, including 13 localized TGCT, 21 diffuse TGCT, and five of unspecified type. CSF1 rearrangements were identified by FISH in 30 cases: 13 translocations, 17 3' deletions. Sequencing confirmed CSF1 breakpoints in 28 cases; in all 28 the breakpoint was found to be downstream of exon 5, replacing or deleting a long 3' UTR containing known miRNA and AU-rich element negative regulatory sequences. We also confirmed the presence of CBL exon 8-9 mutations in six of 21 cases. In conclusion, TGCT in our large cohort were characterized by variable alterations, all of which led to truncation of the 3' end of CSF1, instead of the COL6A3-CSF1 fusions previously reported in some TGCTs. The diversity of fusion partners but consistent integrity of CSF1 functional domains encoded by exons 1-5 support a hypothesis that CSF1 overexpression results from transcription of a truncated form of CSF1 lacking 3' negative regulatory sequences. The presence of CBL mutations affecting the linker and RING finger domain suggests an alternative mechanism for increased CSF1/CSF1R signaling in some cases.
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http://dx.doi.org/10.1002/gcc.22807DOI Listing
February 2020

A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples.

BMC Genomics 2017 06 5;18(1):442. Epub 2017 Jun 5.

Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.

Background: RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A) + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives.

Methods: We compared for the first time three commercial Illumina library preparation kits (TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access) as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels (intact, degraded, and highly degraded).

Results: We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results (R  > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input.

Conclusions: We found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. We also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples.
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http://dx.doi.org/10.1186/s12864-017-3827-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460543PMC
June 2017

Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families.

Am J Hum Genet 2017 May 27;100(5):737-750. Epub 2017 Apr 27.

Division of Human Genetics, School of Pathology and the Sydney Brenner Institute for Molecular Bioscience, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa. Electronic address:

Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.
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http://dx.doi.org/10.1016/j.ajhg.2017.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420352PMC
May 2017

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors.

Nat Commun 2017 02 10;8:14262. Epub 2017 Feb 10.

Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics/Otto Warburg Laboratory Gene Regulation and Systems Biology of cancer, Ihnestrasse 73, D-14195 Berlin, Germany.

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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http://dx.doi.org/10.1038/ncomms14262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309787PMC
February 2017

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Cell 2016 11;167(5):1398-1414.e24

Department of Human Genetics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK; Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Long Road, Cambridge CB2 0PT, UK; British Heart Foundation Centre of Excellence, Division of Cardiovascular Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK; The National Institute for Health Research Blood and Transplant Unit (NIHR BTRU) in Donor Health and Genomics at the University of Cambridge, Strangeways Research Laboratory, University of Cambridge, Wort's Causeway, Cambridge CB1 8RN, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2016.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119954PMC
November 2016

Dissecting the Effect of Genetic Variation on the Hepatic Expression of Drug Disposition Genes across the Collaborative Cross Mouse Strains.

Front Genet 2016 5;7:172. Epub 2016 Oct 5.

Department of Cell Research and Immunology, Faculty of Life Sciences, Tel-Aviv University Tel-Aviv, Israel.

A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC) mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs). By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences.
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http://dx.doi.org/10.3389/fgene.2016.00172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050206PMC
October 2016

Antimalarial Activity of KAF156 in Falciparum and Vivax Malaria.

N Engl J Med 2016 09;375(12):1152-60

From the Mahidol-Oxford Tropical Medicine Research Unit (MORU) (N.J.W.) and the Department of Clinical Tropical Medicine (B.H., S.P., P.J.), Faculty of Tropical Medicine, Mahidol University, Bangkok, Shoklo Malaria Research Unit (SMRU), Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot (F.N., A.P.P.), Phusing Hospital, 83/1 Tambon Huay Tikchu, Phusing District, Srisaket (K.C.), and Mae Ramat District Hospital, Mae Ramat District, Tak (C.U.) - all in Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom (N.J.W., F.N.); National Institute for Malariology, Parasitology and Entomology, Tu Liem District, Hanoi (D.T.T.); Novartis Institutes for BioMedical Research (M.S.C., M.S., D.W.) and Novartis Pharma (B.M.) - both in Basel, Switzerland; Novartis Institutes for BioMedical Research (Y.F., R.Z.) and Novartis Pharma (China) (X.X.), Shanghai, and Novartis Institutes for BioMedical Research, Beijing (R.L.) - all in China; Novartis Institute for Tropical Diseases, Singapore (T.T.D., F.J.L.); and Novartis Institutes for BioMedical Research, Cambridge, MA (P.P.).

Background: KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites.

Methods: We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a separate cohort of patients with falciparum malaria who received a single dose (800 mg).

Results: Median parasite clearance times were 45 hours (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 hours (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 hours (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 hours. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts.

Conclusions: KAF156 showed antimalarial activity without evident safety concerns in a small number of adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and others; ClinicalTrials.gov number, NCT01753323 .).
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http://dx.doi.org/10.1056/NEJMoa1602250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142602PMC
September 2016

Novel mutation in the CHST6 gene causes macular corneal dystrophy in a black South African family.

BMC Med Genet 2016 07 20;17(1):47. Epub 2016 Jul 20.

Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, 2050, Johannesburg, Gauteng, South Africa.

Background: Macular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters.

Methods: A multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact.

Results: Ophthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity.

Conclusion: We identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.
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http://dx.doi.org/10.1186/s12881-016-0308-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955246PMC
July 2016

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

Nat Genet 2015 Sep 27;47(9):1020-1029. Epub 2015 Jul 27.

Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany.

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.
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http://dx.doi.org/10.1038/ng.3362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603357PMC
September 2015

Comparative analysis and modeling of the severity of steatohepatitis in DDC-treated mouse strains.

PLoS One 2014 27;9(10):e111006. Epub 2014 Oct 27.

Max Planck Institute for Molecular Genetics, Department Vertebrate Genomics, Berlin, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210132PMC
June 2015

Influence of RNA extraction methods and library selection schemes on RNA-seq data.

BMC Genomics 2014 Aug 11;15:675. Epub 2014 Aug 11.

Max Planck Institute for Molecular Genetics, Ihnestr, 63-73, Berlin 14195, Germany.

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.
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http://dx.doi.org/10.1186/1471-2164-15-675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148917PMC
August 2014

Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing.

Nature 2014 Jun 18;510(7506):537-41. Epub 2014 May 18.

1] Division of Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany [2] Heidelberg Center for Personalised Oncology (DKFZ-HIPO), Im Neuenheimer Feld 280, Heidelberg 69120, Germany.

Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
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http://dx.doi.org/10.1038/nature13268DOI Listing
June 2014

Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation.

Sci Signal 2014 May 13;7(325):rs3. Epub 2014 May 13.

The David and Inez Myers Laboratory for Cancer Research, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

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.
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http://dx.doi.org/10.1126/scisignal.2005032DOI Listing
May 2014

Hypermutation of the inactive X chromosome is a frequent event in cancer.

Cell 2013 Oct 17;155(3):567-81. Epub 2013 Oct 17.

Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

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.
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http://dx.doi.org/10.1016/j.cell.2013.09.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898475PMC
October 2013

Transcriptome and genome sequencing uncovers functional variation in humans.

Nature 2013 Sep 15;501(7468):506-11. Epub 2013 Sep 15.

Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, Switzerland.

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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http://dx.doi.org/10.1038/nature12531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918453PMC
September 2013

Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma.

Nat Genet 2013 Aug 30;45(8):927-32. Epub 2013 Jun 30.

Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. 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 new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.
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http://dx.doi.org/10.1038/ng.2682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951336PMC
August 2013

Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins.

Cell Rep 2013 Apr 18;3(4):1321-33. Epub 2013 Apr 18.

Foundation FIRC Institute of Molecular Oncology at the IFOM-IEO Campus, via Adamello 16, 20139 Milan, Italy.

The interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.
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http://dx.doi.org/10.1016/j.celrep.2013.03.029DOI Listing
April 2013

Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer.

Cancer Cell 2013 Feb;23(2):159-70

Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany.

Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.
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http://dx.doi.org/10.1016/j.ccr.2013.01.002DOI Listing
February 2013

Dissecting the genomic complexity underlying medulloblastoma.

Nature 2012 Aug;488(7409):100-5

Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany.

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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http://dx.doi.org/10.1038/nature11284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662966PMC
August 2012

A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods.

Biochem Biophys Res Commun 2012 Jun 15;422(4):643-6. Epub 2012 May 15.

Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany.

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.
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http://dx.doi.org/10.1016/j.bbrc.2012.05.043DOI Listing
June 2012

A strand-specific library preparation protocol for RNA sequencing.

Methods Enzymol 2011 ;500:79-98

Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestrasse 63-73, Berlin, Germany.

The analysis of transcriptome, which was over the past decade based mostly on microarray technologies, is now being superseded by so-called next generation sequencing (NGS) systems that changed the way to explore entire transcriptome. RNA sequencing (RNA-Seq), one application of NGS, is a powerful tool, providing information not only about the expression level of genes but also further about the structure of transcripts as it enables to unequivocally identify splicing events, RNA editing products, and mutations in expressed coding sequences within a single experiment. Herein, we describe step by step the deoxy-UTP (dUTP) strand-marking protocol [Parkhomchuk, D., Borodina, T., Amstislavskiy, V., Banaru, M., Hallen, L., Krobitsch, S., Lehrach, H., Soldatov, A. (2009). Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res.37(18), e123], which has been recently reviewed as the leading protocol for strand-specific RNA-Seq library preparation [Levin, J. Z., Yassour, M., Adiconis, X., Nusbaum, C., Thompson, D. A., Friedman, N., Gnirke, A., Regev, A. (2009). Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods7(9), 709-715]. The procedure starts with the isolation of the polyA fraction (mRNA) within a pool of total RNA, followed by its fragmentation. Then double-stranded (ds) cDNA synthesis is performed with the incorporation of dUTP in the second strand. The ds cDNA fragments are further processed following a standard sequencing library preparation scheme tailored for the Illumina sequencing platform: end polishing, A-tailing, adapter ligation, and size selection. Prior to final amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase (UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing.
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http://dx.doi.org/10.1016/B978-0-12-385118-5.00005-0DOI Listing
January 2012

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

J Biol Chem 2011 Jul 9;286(26):23521-32. Epub 2011 May 9.

Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.
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http://dx.doi.org/10.1074/jbc.M111.220178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123115PMC
July 2011

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

PLoS Biol 2011 Jan 18;9(1):e1000582. Epub 2011 Jan 18.

Telethon Institute of Genetics and Medicine, Naples, Italy.

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.
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http://dx.doi.org/10.1371/journal.pbio.1000582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022534PMC
January 2011

The genome of a songbird.

Nature 2010 Apr;464(7289):757-62

The Genome Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park Avenue, St Louis, Missouri 63108, USA.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.
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http://dx.doi.org/10.1038/nature08819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187626PMC
April 2010

Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments.

Nucleic Acids Res 2010 Jun 11;38(10):e112. Epub 2010 Feb 11.

Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Ihnestr 73, 14195 Berlin, Germany.

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/.
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http://dx.doi.org/10.1093/nar/gkq041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879520PMC
June 2010

A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome.

Science 2008 Aug 3;321(5891):956-60. Epub 2008 Jul 3.

Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.

The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.
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http://dx.doi.org/10.1126/science.1160342DOI Listing
August 2008

Gene expression variation in Down's syndrome mice allows prioritization of candidate genes.

Genome Biol 2007 ;8(5):R91

Max Planck Institute for Molecular Genetics, Ihnestr,63/73, 14195, Berlin, Germany.

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.
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http://dx.doi.org/10.1186/gb-2007-8-5-r91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1929163PMC
September 2007

Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

Biomed Microdevices 2007 Jun;9(3):307-14

Max-Planck-Institute for Molecular Genetics, Department Vertebrate Genomics, Ihnestrasse 73, 14195 Berlin-Dahlem, Germany.

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.
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http://dx.doi.org/10.1007/s10544-006-9034-2DOI Listing
June 2007

Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome.

Genome Res 2004 Jul;14(7):1258-67

Max Planck Institute for Molecular Genetics, D-14195, Berlin, Germany.

Human trisomy 21, which results in Down syndrome (DS), is one of the most complicated congenital genetic anomalies compatible with life, yet little is known about the molecular basis of DS. It is generally accepted that chromosome 21 (Chr21) transcripts are overexpressed by about 50% in cells with an extra copy of this chromosome. However, this assumption is difficult to test in humans due to limited access to tissues, and direct support for this idea is available for only a few Chr21 genes or in a limited number of tissues. The Ts65Dn mouse is widely used as a model for studies of DS because it is at dosage imbalance for the orthologs of about half of the 284 Chr21 genes. Ts65Dn mice have several features that directly parallel developmental anomalies of DS. Here we compared the expression of 136 mouse orthologs of Chr21 genes in nine tissues of the trisomic and euploid mice. Nearly all of the 77 genes which are at dosage imbalance in Ts65Dn showed increased transcript levels in the tested tissues, providing direct support for a simple model of increased transcription proportional to the gene copy number. However, several genes escaped this rule, suggesting that they may be controlled by additional tissue-specific regulatory mechanisms revealed in the trisomic situation.
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http://dx.doi.org/10.1101/gr.1951304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC442140PMC
July 2004