Paean: A parallel transcriptome quantification tool combining gene expression and alternative splicing events using GPU

Abstract Alternative splicing is an important post-translational change that alters gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Here we have analyzed alternative splicing in myeloma using high throughput RNA-seq. Our analytic pipeline for RNA-seq data used in this investigation not only provides information on expression levels for genes, but also provides information on the expression of known splice variants of genes (isoforms), and can identify novel exon level events across individuals (i.e. exon skipping events). We conducted a study of 328 newly-diagnosed patients with multiple myeloma treated homogeneously with novel agent combination containting lenalidomide, bortezomib and dexamethsone with or without high-dose melphalan followed by lenalidomide maintenance in the IFM/DFCI study. RNA isolated from purified CD138+ MM cells collected at the time of diagnosis and from 18 normal donor plasma cells were processed by RNA-seq (100 million paired end reads on Illumina HiSeq) and analyzed using a custom computational and statistical pipeline. Following read alignment to hg19, we utilized RSEM to quantify both gene-level and isoform-level expression of known ENSEMBL transcripts. We then implemented a novel testing approach based on compositional regression to discover genes that show significant isoform switching between the 328 MM samples and 18 Normal Plasma Cell (NPC) samples from healthy donors. Using various programs and their modifications, we also identified novel alternative splicing events, such as exon skipping and mutually exclusive exon usage, among others. Patient data for MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data. We observed over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. A number of previously characterized genes including MYCL1 (adj. p = 0.0014) and CCND3 (adj. p = 0.0013), and MAP kinase-related genes (MAP3K8, MAPKAPK2, MAPKAPK3, MAP4K4) exhibited significant isoform switching compared to normal, in addition to some not well characterized genes. Genes showing the greatest magnitude of isoform switching include MEFV (adj. p = 2.7 x 10-5), showing a two fold change in the relative major isoform abundance compared to normal, and has been previously shown to have a role in lymphoid neoplasms. We applied hierarchical clustering to the isoforms showing significant changes in isoform-switching and identified 4 distinct clusters, which are currently being investigated for correlation with clinical subtypes of MM. Exon level analyses of alternative splicing events, such as exon skipping, are currently underway. Clinical data including MM characteristics, cytogenetics, FISH and survival outcomes was available for a subset of 265 patients. We found that 109 genes showed significant isoform switching between t(4;14) and non-t(4;14) patients, such as CD44 (adj. p =1.8 x 10-6) and WHSC1 (adj. p =5.1 x 10-28). Comparing del17p (28 in total) and non del17p patients, we found no significant splicing changes after multiple testing adjustment. Of these genes, only a subset (40%) were shown to be differentially expressed in terms of total gene expression, suggesting the importance of examining alternative splicing events in addition to total gene expression. With respect to treatment response, we compared the expression of gene isoforms between patients achieving complete response (CR) versus others and identified 38 isoforms associated with response to treatment (adj. p value < 0.05), with SEPT9, SLC2A5, and UBX6 having the strongest associations (adj. p-value < 3 x 10-4). Using a univariate cox regression model, 4 spliced isoforms relating to 3 genes were identified as having significant correlation with event-free survival (EFS) (FDR-adjusted cox p value < 0.05). We are in the process of now integrating the gene expression data with altered splicing data to develop an integrated survival model. In summary, this study highlights the significant frequency, biological and clinical importance of alternative splicing in MM and points to the need for evaluation of not only the expression level of genes but also post-translational modifications. The genes identified here are important targets for therapy as well as possible immune modulation. Disclosures Moreau: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Data for the gene expression profiling and alternative splicing events during the chondrogenic differentiation of human cartilage endplate-derived stem cells under hypoxia

Data in Brief ◽

10.1016/j.dib.2016.04.037 ◽

2016 ◽

Vol 7 ◽

pp. 1438-1442 ◽

Cited By ~ 2

Author(s):

Yuan Yao ◽

Jin Shang ◽

Weilin Song ◽

Qiyue Deng ◽

Huan Liu ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Alternative Splicing ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Chondrogenic Differentiation ◽

Human Cartilage ◽

Cartilage Endplate ◽

Alternative Splicing Events

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Computer Analysis of Glioma Transcriptome Profiling: Alternative Splicing Events

Journal of Integrative Bioinformatics ◽

10.1515/jib-2017-0022 ◽

2017 ◽

Vol 14 (3) ◽

Cited By ~ 2

Author(s):

Vladimir N. Babenko ◽

Natalya V. Gubanova ◽

Anatoly O. Bragin ◽

Irina V. Chadaeva ◽

Gennady V. Vasiliev ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Alternative Splicing ◽

Expression Profiles ◽

Rna Extraction ◽

Transcriptome Profiling ◽

Laboratory Animals ◽

Normal Brain ◽

Computer Tools ◽

Alternative Splicing Events

AbstractHere we present the analysis of alternative splicing events on an example of glioblastoma cell culture samples using a set of computer tools in combination with database integration. The gene expression profiles of glioblastoma were obtained from cell culture samples of primary glioblastoma which were isolated and processed for RNA extraction. Transcriptome profiling of normal brain samples and glioblastoma were done by Illumina sequencing. The significant differentially expressed exon-level probes and their corresponding genes were identified using a combination of the splicing index method. Previous studies indicated that tumor-specific alternative splicing is important in the regulation of gene expression and corresponding protein functions during cancer development. Multiple alternative splicing transcripts have been identified as progression markers, including generalized splicing abnormalities and tumor- and stage-specific events. We used a set of computer tools which were recently applied to analysis of gene expression in laboratory animals to study differential splicing events. We found 69 transcripts that are differentially alternatively spliced. Three cancer-associated genes were considered in detail, in particular: APP (amyloid beta precursor protein), CASC4 (cancer susceptibility candidate 4) and TP53. Such alternative splicing opens new perspectives for cancer research.

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Complementary feature selection from alternative splicing events and gene expression for phenotype prediction

Bioinformatics ◽

10.1093/bioinformatics/btw430 ◽

2016 ◽

Vol 32 (17) ◽

pp. i421-i429 ◽

Cited By ~ 6

Author(s):

Charles J. Labuzzetta ◽

Margaret L. Antonio ◽

Patricia M. Watson ◽

Robert C. Wilson ◽

Lauren A. Laboissonniere ◽

...

Keyword(s):

Gene Expression ◽

Feature Selection ◽

Alternative Splicing ◽

Phenotype Prediction ◽

Alternative Splicing Events

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A Combined Survival Model Integrating Gene Expression and Alternative Splicing Events Provides Higher Predicative Power for Risk Stratification

Blood ◽

10.1182/blood.v116.21.1929.1929 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1929-1929

Author(s):

Parantu K Shah ◽

Hervé Avet-Loiseau ◽

Stephane Minvielle ◽

Samir B. Amin ◽

Florence Magrangeas ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Risk Stratification ◽

Cell Lines ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Differential Splicing ◽

Combined Model ◽

Healthy Donors ◽

Alternative Splicing Events

Abstract Abstract 1929 Considerable efforts have been spent evaluating impact of global gene expression profile on clinical outcome. Although significant correlation has been described between outcome and expressed gene signature, the overall predictability of such models have reached a plateau. Biologically this is expected as gene function is modulated at multiple levels. Besides the change in level of expression, post-transcriptional changes such as alternate splicing alter specificity of gene function and may affect the eventual outcome. Although various genes have normal alternate spliced form, dysregulation of alternative splicing that alters protein function has been implicated in number of disease processes including cancer. We have observed significant level of dysregulated splicing events in multiple myeloma (MM). We hypothesize that a combined model that includes dysregulated splicing events, besides level of expressed genes, may provide superior survival model in MM. To develop a combined model we have hybridized RNA isolated from CD138+ purified MM cells collected at the time of diagnosis from 170 newly-diagnosed patients treated homogeneously in tandem transplantation IFM trials, 23 MM cell lines and 6 Healthy donors on Affymetrix Exon 1.0 ST GeneChip arrays. Exon array not only provides an accurate measure of expression levels for genes, but also allows simultaneous identification of alternative splicing events. Pre-processing and normalization methods in aroma, affymetrix and robust multichip analysis model in FIRMA, followed by t-tests with Benjamini-Hochberg multiple hypothesis corrections were used respectively to identify differential expression and alternative splicing. We identified 1454 differentially expressed genes and 759 differential splicing events between healthy donors and MM patients, and 5476 differentially expressed genes and 4012 differential splicing events between healthy donors and MM cell lines. There are 1071 differentially expressed genes and 286 alternative spliced exons shared between MM samples and MM cell lines. Univariate survival analysis using FIRMA scores of exons identified a total of 89 genes with more than 10 alternative splicing events between healthy donors and MM patients associated with survival with Cox proportional hazard model and log-rank tests. We have now built 3 different survival models considering: 1) gene expression only, 2) alternative splicing only, and 3) a combined model integrating gene expression and alternative splicing events. We utilized refined regularized variable selection methods to handle these high-dimensional feature space. Our analysis suggests that composite model using gene expression and alternative splicing information performs significantly better than the gene expression only model in identifying high-risk patients, when the data were divided in median or quartiles. Specifically, the difference in overall survival is 32.6 months to 38.5 months using the median survival, and 18 months vs 23 months for median event free survival. We are currently in the process of validating the combined model. Our data suggests the need for inclusion of modifiers of transcriptome to develop a comprehensive model that will have higher predicative power for risk stratification as well as for selection of therapeutic intervention. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

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A Dynamic Alternative Splicing Program Regulates Gene Expression In A Differentiation Stage-Specific Manner During Terminal Erythropoiesis

Blood ◽

10.1182/blood.v122.21.3413.3413 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3413-3413 ◽

Cited By ~ 1

Author(s):

Harold Pimentel ◽

Marilyn Parra ◽

Jie Li ◽

Sherry Gee ◽

Dana Ghanem ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Globin Gene ◽

Structure And Function ◽

Rna Seq ◽

Arginine Residues ◽

Alternative Splicing Events ◽

And Function

Abstract Spatio-temporal regulation of switches in alternative pre-mRNA splicing modulate exon usage, critically remodeling the transcriptome during development and differentiation of many tissues, while aberrant regulation of alternative splicing disrupts these processes and plays a role in numerous human diseases. Recently, the discovery of splicing factor mutations in myelodysplasia has increased interest in splicing regulation in hematology. Previously, a functionally critical erythroid splicing switch in protein 4.1 pre-mRNA has been reported, in which activation of alternative exon 16 splicing in late erythroblasts is required for assembly of a mechanically stable red cell membrane. To explore globally the landscape of important alternative splicing events in the erythroid lineage, we applied RNA-seq analysis to five highly FACS-purified populations of human erythroblasts, cultured from CD34+ cord blood progenitors, representing proerythroblasts, early and late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. Alternative splicing events predicted by computational analysis were filtered to remove low expression genes and low frequency splicing events, to derive a list of >3000 ‘major’ alternative splicing events of potential importance in erythroid biology. Many of these were validated by inspection of RNA-seq reads mapped on the human genome, and/or by RT-PCR analysis. In this unique differentiation system we found an extensive and dynamic alternative splicing program enriched in genes that function in cell cycle regulation, organelle organization, chromatin structure and function, and RNA processing. For example, we identified alternative splicing events in ∼25 genes encoding chromatin modifying enzymes that methylate, demethylate, or acetylate specific lysine or arginine residues in histones; in transcription modulators such as ATRX and BCL11A that regulate normal globin gene expression; and in ∼50 RNA binding proteins with various roles in post-transcriptional gene regulation. Comparison of PSI (percent spliced in) values across the differentiation series revealed that dozens of alternative exons exhibit substantial switches in splicing efficiency during terminal erythropoiesis. The majority of splicing switches occur in late-stage polychromatophilic and orthochromatophilic erythroblasts, temporally correlated with changes in transcript abundance for many splicing factors and with substantial cell remodeling prior to enucleation. One of the biggest switches in late erythroblasts involves inclusion of a 35nt exon in the NDEL1 (nuclear distribution factor E-homolog-like1) gene, which alters C-terminal structure of a protein that functions in nuclear migration and nucleokinesis in nonerythroid cells and may have a role in erythroblast enucleation. Most of the regulated splicing events insert or delete sequences predicted to modulate protein structure and function in late erythroblasts. However, a subset of altered splicing events have a different effect on gene expression by introducing premature translation termination codons (PTCs), leading us to hypothesize that alternative splicing-coupled nonsense-mediated-decay (AS-NMD) contributes to stage-specific down-regulation of numerous erythroid transcripts. Consistent with such a model, most genes that up-regulate PTC exons in late erythroblasts exhibit reduction in overall expression levels, and inhibition of NMD increases the apparent expression of PTC isoforms. In contrast, genes that up-regulate coding exons are not preferentially down-regulated in late erythroblasts. We conclude that a dynamically regulated alternative splicing program in terminally differentiating erythroblasts plays a major post-transcriptional role in shaping gene expression as the cells transition from proliferation to differentiation, ensuring synthesis of the appropriate constellation of proteins as the cells prepare for enucleation and production of mature red cells. Disclosures: No relevant conflicts of interest to declare.

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Alternative Splicing Events Is Not a Key Event for Gene Expression Regulation in Uremia

PLoS ONE ◽

10.1371/journal.pone.0082702 ◽

2013 ◽

Vol 8 (12) ◽

pp. e82702

Author(s):

Marion Sallée ◽

Michel Fontès ◽

Laurence Louis ◽

Claire Cérini ◽

Philippe Brunet ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Gene Expression Regulation ◽

Expression Regulation ◽

Alternative Splicing Events

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Regulation of gene expression through production of unstable mRNA isoforms

Biochemical Society Transactions ◽

10.1042/bst20140102 ◽

2014 ◽

Vol 42 (4) ◽

pp. 1196-1205 ◽

Cited By ~ 13

Author(s):

Christopher R. Sibley

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Regulation Of Gene Expression ◽

Present Review ◽

Mrna Isoforms ◽

Control Of Gene Expression ◽

Nonsense Mediated Decay ◽

Alternative Splicing Events

Alternative splicing is universally accredited for expanding the information encoded within the transcriptome. In recent years, several tightly regulated alternative splicing events have been reported which do not lead to generation of protein products, but lead to unstable mRNA isoforms. Instead these transcripts are targets for NMD (nonsense-mediated decay) or retained in the nucleus and degraded. In the present review I discuss the regulation of these events, and how many have been implicated in control of gene expression that is instrumental to a number of developmental paradigms. I further discuss their relevance to disease settings and conclude by highlighting technologies that will aid identification of more candidate events in future.

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