Matthias Scherf

A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome

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.

Nitric oxide-responsive genes and promoters in Arabidopsis thaliana: a bioinformatics approach

Due to its high reactivity and its ability to diffuse and permeate the cell membrane, nitric oxide (NO) and its exchangeable redox-activated species are unique biological messengers in animals and in plants. Although an increasing number of reports indicate that NO is an essential molecule in several physiological processes, there is not a clear picture of its method of action. Studies on the transcriptional changes induced by NO permitted identification of genes involved in different functional processes such as signal transduction, defence and cell death, transport, basic metabolism, and reactive oxygen species (ROS) production and degradation. The co-expression of these genes can be explained by the co-operation of a set of transcription factors that bind a common region in the promoter of the regulated genes. The present report describes the search for a common transcription factor-binding site (TFBS) in promoter regions of NO-regulated genes, based on microarray analyses. Using Genomatix Gene2Promotor and MatInspector, eight families of TFBSs were found to occur at least 15% more often in the promoter regions of the responsive genes in comparison with the promoter regions of 28,447 Arabidopsis control genes. Most of these TFBSs, such as ocs element-like sequences and WRKY, have already been reported to be involved in particular stress responses. Furthermore, the promoter regions of genes involved in jasmonic acid (JA) biosynthesis were analysed for a common TFBS module, since some genes responsible for JA biosynthesis are induced by NO, and an interaction between NO and JA signalling has already been described.

Experimental data of a single promoter can be used for in silico detection of genes with related regulation in the absence of sequence similarity

Abstract. Gene expression is presently a major focus in genome analysis, and the experimental data on regulatory mechanisms and functional transcription factor binding sites are steadily growing. However, the annotation of transcriptional regulation of sequences cannot keep pace with the exponential growth of sequence databases. Employing detailed experimental data of a single promoter or enhancer to predict genes with similar regulation would provide a powerful method to link the literature about transcriptional regulation and sequence databases. To this end, we used information on individual functional transcription factor binding sites to compose in silico promoter and enhancer models of muscle-specific genes and to analyze the rodents section of EMBL with these models. Exhaustive evaluation of all hits revealed every second to third match to be a muscle-associated gene. Moreover, functionally related regulatory regions were detected by our model-based approach even in the absence of sequence similarity. We believe that this new approach is a substanial extension to database analysis by BLAST or FASTA, which are restricted to sequence similarity.

A novel genomic alteration of LSAMP associates with aggressive prostate cancer in African American men

Evaluation of cancer genomes in global context is of great interest in light of changing ethnic distribution of the world population. We focused our study on men of African ancestry because of their disproportionately higher rate of prostate cancer (CaP) incidence and mortality. We present a systematic whole genome analyses, revealing alterations that differentiate African American (AA) and Caucasian American (CA) CaP genomes. We discovered a recurrent deletion on chromosome 3q13.31 centering on the LSAMP locus that was prevalent in tumors from AA men (cumulative analyses of 435 patients: whole genome sequence, 14; FISH evaluations, 101; and SNP array, 320 patients). Notably, carriers of this deletion experienced more rapid disease progression. In contrast, PTEN and ERG common driver alterations in CaP were significantly lower in AA prostate tumors compared to prostate tumors from CA. Moreover, the frequency of inter-chromosomal rearrangements was significantly higher in AA than CA tumors. These findings reveal differentially distributed somatic mutations in CaP across ancestral groups, which have implications for precision medicine strategies.

Detecting and visualizing gene fusions

In recent years, gene fusions have gained significant recognition as biomarkers. They can assist treatment decisions, are seldom found in normal tissue and are detectable through Next-generation sequencing (NGS) of the transcriptome (RNA-seq). To transform the data provided by the sequencer into robust gene fusion detection several analysis steps are needed. Usually the first step is to map the sequenced transcript fragments (RNA-seq) to a reference genome. One standard application of this approach is to estimate expression and detect variants within known genes, e.g. SNPs and indels. In case of gene fusions, however, completely novel gene structures have to be detected. Here, we describe the detection of such gene fusion events based on our comprehensive transcript annotation (ElDorado). To demonstrate the utility of our approach, we extract gene fusion candidates from eight breast cancer cell lines, which we compare to experimentally verified gene fusions. We discuss several gene fusion events, like BCAS3-BCAS4 that was only detected in the breast cancer cell line MCF7. As supporting evidence we show that gene fusions occur more frequently in copy number enriched regions (CNV analysis). In addition, we present the Transcriptome Viewer (TViewer) a tool that allows to interactively visualize gene fusions. Finally, we support detected gene fusions through literature mining based annotations and network analyses. In conclusion, we present a platform that allows detecting gene fusions and supporting them through literature knowledge as well as rich visualization capabilities. This enables scientists to better understand molecular processes, biological functions and disease associations, which will ultimately lead to better biomedical knowledge for the development of biomarkers for diagnostics and therapies.

BiblioSphere — Hypothesis Generation in Regulatory Network Analysis

With its microarray analysis strategy BiblioSphere introduced a new analysis concept for the interpretation of microarray results. The first level of analysis integrates multiple complementary data sources, to compile a network that covers the entire knowledge and data available for the biological entities in the input data set to enable the interactive analysis of the data. A broad range of filters together with ranking techniques are available as tools to enable the sub grouping of entities and relations to focus the analysis to the user’s interest.

Abstract 140: LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men

INTRODUCTION: Disproportionately higher rates of prostate cancer (CaP) incidence and mortality have been reported among African American (AA) men. Although oncogenic TMPRSS2-ERG gene fusion and deletion of the PTEN tumor suppressor gene are established cancer driver gene alterations in CaP, they are known to be more prevalent among men of European ancestry. By utilizing carefully annotated specimens, this study focused on the discovery of recurrent genomic alterations in CaP of AA men in comparison to Caucasian Americans (CA). METHODS: Genomic DNA from clinically localized primary prostate tumors (Gleason 6 or 7 with primary pattern 3) and matched peripheral blood lymphocytes of seven AA and seven CA patients, were analyzed by paired-end sequencing on Illumina Genome Analyzer IIx to a depth of 30x. Following alignment to reference genome, somatic alterations on tumor DNA that include single nucleotide variants (SNVs), insertion and deletions (Indels), structural variations, copy number variations, and inter- and intra-chromosomal translocations of tumor DNA sequence were identified. To confirm prevalent genomic deletions we performed FISH analysis on a tissue microarray constructed from 42 AA and 59 CA tumor and normal samples of an independent cohort. Frequently deleted loci were further validated by analysis of TCGA CaP SNP array data from 41 AA and 279 CA prostate tumors. RESULTS: A comparative evaluation of whole genome sequences of AA and CA CaP revealed a prevalent deletion of the LSAMP locus of chromosome 3q13.31 in AA CaP. These observations were confirmed by SNP array and FISH assays in independent cohorts of specimens. AA CaP patients with LSAMP deletion showed rapid disease progression. In contrast to higher frequency of LSAMP deletion, significantly lower frequencies of PTEN and ERG alterations were noted in CaP of AA men. Furthermore, CaP genomes of AA men displayed a higher rate of inter-chromosomal rearrangements than those from CA men. CONCLUSIONS: We highlight distinct features of AA and CA CaP genomes including common CaP driver genes (TMPRSS2- ERG, PTEN) and define a novel recurrent deletion of the LSAMP locus. This study underscores the need for careful evaluations of cancer genomes in underrepresented populations in the global context with implications for precision medicine strategies. Citation Format: Albert Dobi, Gyorgy Petrovics, Hua Li, Shyh-Han Tan, Tanja Stumpel, Denise Young, Shilpa Katta, Qiyuan Li, Kai Ying, Bernward Klocke, Lakshmi Ravindranath, Indu Kohaar, Yongmei Chen, Dezső Ribli, Korbinian Grote, Hau Zou, Joseph Cheng, Clifton L. Dalgard, Shimin Zhang, Istvan Csabai, Jacob Kagan, David Takeda, Massimo Loda, Sudhir Srivastava, Matthias Scherf, Martin Seifert, Timo Gaiser, David G. McLeod, Zoltan Szallasi, Reinhard Ebner, Thomas Werner, Isabell A. Sesterhenn, Matthew Freedman, Shiv Srivastava. LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 140.