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Find video protocols related to scientific articles indexed in Pubmed.
RVboost: RNA-seq variants prioritization using a boosting method.
Bioinformatics
PUBLISHED: 08-27-2014
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RNA-seq has become the method of choice to quantify genes and exons, discover novel transcripts and detect fusion genes. However, reliable variant identification from RNA-seq data remains challenging because of the complexities of the transcriptome, the challenges of accurately mapping exon boundary spanning reads and the bias introduced during the sequencing library preparation.
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Analysis of COQ2 gene in multiple system atrophy.
Mol Neurodegener
PUBLISHED: 08-26-2014
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Loss of function COQ2 mutations results in primary CoQ10 deficiency. Recently, recessive mutations of the COQ2 gene have been identified in two unrelated Japanese families with multiple system atrophy (MSA). It has also been proposed that specific heterozygous variants in the COQ2 gene may confer susceptibility to sporadic MSA. To assess the frequency of COQ2 variants in patients with MSA, we sequenced the entire coding region and investigated all exonic copy number variants of the COQ2 gene in 97 pathologically-confirmed and 58 clinically-diagnosed MSA patients from the United States.
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Genome-wide association study identifies multiple susceptibility loci for diffuse large B cell lymphoma.
James R Cerhan, Sonja I Berndt, Joseph Vijai, Hervé Ghesquières, James McKay, Sophia S Wang, Zhaoming Wang, Meredith Yeager, Lucia Conde, Paul I W de Bakker, Alexandra Nieters, David Cox, Laurie Burdett, Alain Monnereau, Christopher R Flowers, Anneclaire J De Roos, Angela R Brooks-Wilson, Qing Lan, Gianluca Severi, Mads Melbye, Jian Gu, Rebecca D Jackson, Eleanor Kane, Lauren R Teras, Mark P Purdue, Claire M Vajdic, John J Spinelli, Graham G Giles, Demetrius Albanes, Rachel S Kelly, Mariagrazia Zucca, Kimberly A Bertrand, Anne Zeleniuch-Jacquotte, Charles Lawrence, Amy Hutchinson, Degui Zhi, Thomas M Habermann, Brian K Link, Anne J Novak, Ahmet Dogan, Yan W Asmann, Mark Liebow, Carrie A Thompson, Stephen M Ansell, Thomas E Witzig, George J Weiner, Amelie S Veron, Diana Zelenika, Hervé Tilly, Corinne Haioun, Thierry Jo Molina, Henrik Hjalgrim, Bengt Glimelius, Hans-Olov Adami, Paige M Bracci, Jacques Riby, Martyn T Smith, Elizabeth A Holly, Wendy Cozen, Patricia Hartge, Lindsay M Morton, Richard K Severson, Lesley F Tinker, Kari E North, Nikolaus Becker, Yolanda Benavente, Paolo Boffetta, Paul Brennan, Lenka Foretova, Marc Maynadié, Anthony Staines, Tracy Lightfoot, Simon Crouch, Alex Smith, Eve Roman, W Ryan Diver, Kenneth Offit, Andrew Zelenetz, Robert J Klein, Danylo J Villano, Tongzhang Zheng, Yawei Zhang, Theodore R Holford, Anne Kricker, Jenny Turner, Melissa C Southey, Jacqueline Clavel, Jarmo Virtamo, Stephanie Weinstein, Elio Riboli, Paolo Vineis, Rudolph Kaaks, Dimitrios Trichopoulos, Roel C H Vermeulen, Heiner Boeing, Anne Tjonneland, Emanuele Angelucci, Simonetta Di Lollo, Marco Rais, Brenda M Birmann, Francine Laden, Edward Giovannucci, Peter Kraft, Jinyan Huang, Baoshan Ma, Yuanqing Ye, Brian C H Chiu, Joshua Sampson, Liming Liang, Ju-Hyun Park, Charles C Chung, Dennis D Weisenburger, Nilanjan Chatterjee, Joseph F Fraumeni, Susan L Slager, Xifeng Wu, Silvia de Sanjosé, Karin E Smedby, Gilles Salles, Christine F Skibola, Nathaniel Rothman, Stephen J Chanock.
Nat. Genet.
PUBLISHED: 06-26-2014
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Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma subtype and is clinically aggressive. To identify genetic susceptibility loci for DLBCL, we conducted a meta-analysis of 3 new genome-wide association studies (GWAS) and 1 previous scan, totaling 3,857 cases and 7,666 controls of European ancestry, with additional genotyping of 9 promising SNPs in 1,359 cases and 4,557 controls. In our multi-stage analysis, five independent SNPs in four loci achieved genome-wide significance marked by rs116446171 at 6p25.3 (EXOC2; P = 2.33 × 10(-21)), rs2523607 at 6p21.33 (HLA-B; P = 2.40 × 10(-10)), rs79480871 at 2p23.3 (NCOA1; P = 4.23 × 10(-8)) and two independent SNPs, rs13255292 and rs4733601, at 8q24.21 (PVT1; P = 9.98 × 10(-13) and 3.63 × 10(-11), respectively). These data provide substantial new evidence for genetic susceptibility to this B cell malignancy and point to pathways involved in immune recognition and immune function in the pathogenesis of DLBCL.
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PatternCNV: a versatile tool for detecting copy number changes from exome sequencing data.
Bioinformatics
PUBLISHED: 05-29-2014
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Exome sequencing (exome-seq) data, which are typically used for calling exonic mutations, have also been utilized in detecting DNA copy number variations (CNVs). Despite the existence of several CNV detection tools, there is still a great need for a sensitive and an accurate CNV-calling algorithm with built-in QC steps, and does not require a paired reference for each sample.
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Recurrent PAX3-MAML3 fusion in biphenotypic sinonasal sarcoma.
Nat. Genet.
PUBLISHED: 04-30-2014
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Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Here we report a recurrent chromosomal translocation in SNS, t(2;4)(q35;q31.1), resulting in a PAX3-MAML3 fusion protein that is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, closely simulating the developmental roles of PAX3.
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MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing.
BMC Bioinformatics
PUBLISHED: 02-22-2014
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Although the costs of next generation sequencing technology have decreased over the past years, there is still a lack of simple-to-use applications, for a comprehensive analysis of RNA sequencing data. There is no one-stop shop for transcriptomic genomics. We have developed MAP-RSeq, a comprehensive computational workflow that can be used for obtaining genomic features from transcriptomic sequencing data, for any genome.
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Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.
PLoS Genet.
PUBLISHED: 02-01-2014
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Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50?350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50?8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.
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RNA Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Reduced Expression of Immune Function Genes in BRAF (V600E) Mutant vs BRAF Wild-Type Papillary Thyroid Carcinoma.
J. Clin. Endocrinol. Metab.
PUBLISHED: 12-02-2013
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Context:The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression.Objective:RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status.Design:BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes.Setting:Referral medical center.Patients:BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes.Results:RNA-Seq identified 560 of 13 t085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1-B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR.Conclusion:BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.
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Novel TRAF1-ALK fusion identified by deep RNA sequencing of anaplastic large cell lymphoma.
Genes Chromosomes Cancer
PUBLISHED: 06-03-2013
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Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high-throughput, "deep" sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1-ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50-year-old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non-NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1-ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1-ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery. © 2013 Wiley Periodicals, Inc.
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Identification of novel variants in colorectal cancer families by high-throughput exome sequencing.
Cancer Epidemiol. Biomarkers Prev.
PUBLISHED: 05-01-2013
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Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of exome sequencing in coding regions may identify novel segregating variants.
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A phase I trial of immunostimulatory CpG 7909 oligodeoxynucleotide and (90) yttrium ibritumomab tiuxetan radioimmunotherapy for relapsed B-cell non-Hodgkin lymphoma.
Am. J. Hematol.
PUBLISHED: 03-27-2013
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Radioimmunotherapy (RIT) for relapsed indolent non-Hodgkin lymphoma produces overall response rates (ORR) of 80% with mostly partial remissions. Synthetic CpG oligonucleotides change the phenotype of malignant B-cells, are immunostimulatory, and can produce responses when injected intratumorally and combined with conventional radiation. In this phase I trial, we tested systemic administration of both CpG and RIT. Eligible patients had biopsy-proven previously treated CD20+ B-cell NHL and met criteria for RIT. Patients received rituximab 250 mg/m(2) days 1,8, and 15; (111) In-ibritumomab tiuxetan days 1, 8; CpG 7909 days 6, 13, 20, 27; and 0.4 mCi/kg of (90) Y-ibritumomab tiuxetan day 15. The doses of CpG 7909 tested were 0.08, 0.16, 0.32 (six patients each) and 0.48 mg/kg (12 patients) IV over 2 hr without dose limiting toxicity. The ORR was 93% (28/30) with 63% (19/30) complete remission (CR); median progression free survival of 42.7 months (95% CI 18-NR); and median duration of response (DR) of 35 months (4.6-76+). Correlative studies demonstrated a decrease in IL10 and TNF?, and an increase in IL1?, in response to therapy. CpG 7909 at a dose of 0.48 mg/kg is safe with standard RIT and produces a high CR rate and long DR; these results warrant confirmation. Am. J. Hematol., 2013. © 2013 Wiley Periodicals, Inc.
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CXCR5 polymorphisms in non-Hodgkin lymphoma risk and prognosis.
Cancer Immunol. Immunother.
PUBLISHED: 03-11-2013
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CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4+ and CD8+ T-cells, and skin-derived migratory dendritic cells. Together with its ligand, CXCL13, CXCR5 is involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. This study evaluated the role of common germline genetic variation in CXCR5 in the risk and prognosis of non-Hodgkin lymphoma (NHL) using a clinic-based study of 1,521 controls and 2,694 NHL cases including 710 chronic lymphocytic leukemia/small lymphocytic lymphoma, 586 diffuse large B-cell lymphoma (DLBCL), 588 follicular lymphoma (FL), 137 mantle cell lymphoma (MCL), 230 marginal zone lymphoma (MZL), and 158 peripheral T-cell lymphoma (PTCL). Of the ten CXCR5 tag SNPs in our study, five were associated with risk of NHL, with rs1790192 having the strongest association (OR 1.19, 95% CI 1.08-1.30; p = 0.0003). This SNP was most strongly associated with the risk of FL (OR 1.44, 95 % CI 1.25-1.66; p = 3.1 × 10(-7)), with a lower degree of association with DLBCL (OR 1.16, 95% CI 1.01-1.33; p = 0.04) and PTCL (OR 1.29, 95 % CI 1.02-1.64; p = 0.04) but no association with the risk of MCL or MZL. For FL patients that were observed as initial disease management, the number of minor alleles of rs1790192 was associated with better event-free survival (HR 0.64; 95% CI 0.47-0.87; p = 0.004). These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, particularly for FL.
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Mapping of the IRF8 gene identifies a 3UTR variant associated with risk of chronic lymphocytic leukemia but not other common non-Hodgkin lymphoma subtypes.
Cancer Epidemiol. Biomarkers Prev.
PUBLISHED: 01-10-2013
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Our genome-wide association study (GWAS) of chronic lymphocytic leukemia (CLL) identified 4 highly correlated intronic variants within the IRF8 gene that were associated with CLL. These results were further supported by a recent meta-analysis of our GWAS with two other GWAS of CLL, supporting the IRF8 gene as a strong candidate for CLL risk.
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Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.
PLoS ONE
PUBLISHED: 01-01-2013
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Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16). Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.
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An Integrated Model of the Transcriptome of HER2-Positive Breast Cancer.
PLoS ONE
PUBLISHED: 01-01-2013
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Our goal in these analyses was to use genomic features from a test set of primary breast tumors to build an integrated transcriptome landscape model that makes relevant hypothetical predictions about the biological and/or clinical behavior of HER2-positive breast cancer. We interrogated RNA-Seq data from benign breast lesions, ER+, triple negative, and HER2-positive tumors to identify 685 differentially expressed genes, 102 alternatively spliced genes, and 303 genes that expressed single nucleotide sequence variants (eSNVs) that were associated with the HER2-positive tumors in our survey panel. These features were integrated into a transcriptome landscape model that identified 12 highly interconnected genomic modules, each of which represents a cellular processes pathway that appears to define the genomic architecture of the HER2-positive tumors in our test set. The generality of the model was confirmed by the observation that several key pathways were enriched in HER2-positive TCGA breast tumors. The ability of this model to make relevant predictions about the biology of breast cancer cells was established by the observation that integrin signaling was linked to lapatinib sensitivity in vitro and strongly associated with risk of relapse in the NCCTG N9831 adjuvant trastuzumab clinical trial dataset. Additional modules from the HER2 transcriptome model, including ubiquitin-mediated proteolysis, TGF-beta signaling, RHO-family GTPase signaling, and M-phase progression, were linked to response to lapatinib and paclitaxel in vitro and/or risk of relapse in the N9831 dataset. These data indicate that an integrated transcriptome landscape model derived from a test set of HER2-positive breast tumors has potential for predicting outcome and for identifying novel potential therapeutic strategies for this breast cancer subtype.
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Impact of library preparation on downstream analysis and interpretation of RNA-Seq data: comparison between Illumina PolyA and NuGEN Ovation protocol.
PLoS ONE
PUBLISHED: 01-01-2013
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The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing.
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TREAT: a bioinformatics tool for variant annotations and visualizations in targeted and exome sequencing data.
Bioinformatics
PUBLISHED: 11-15-2011
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TREAT (Targeted RE-sequencing Annotation Tool) is a tool for facile navigation and mining of the variants from both targeted resequencing and whole exome sequencing. It provides a rich integration of publicly available as well as in-house developed annotations and visualizations for variants, variant-hosting genes and host-gene pathways.
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c-Myc regulates self-renewal in bronchoalveolar stem cells.
PLoS ONE
PUBLISHED: 07-25-2011
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Bronchoalveolar stem cells (BASCs) located in the bronchoalveolar duct junction are thought to regenerate both bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. The mechanisms directing self-renewal in BASCs are poorly understood.
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Meta-analysis of 8q24 for seven cancers reveals a locus between NOV and ENPP2 associated with cancer development.
BMC Med. Genet.
PUBLISHED: 07-14-2011
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Human chromosomal region 8q24 contains several genes which could be functionally related to cancer, including the proto-oncogene c-MYC. However, the abundance of associations around 128 Mb on chromosome 8 could mask the appearance of a weaker, but important, association elsewhere on 8q24.
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High-resolution genomic mapping reveals consistent amplification of the fibroblast growth factor receptor substrate 2 gene in well-differentiated and dedifferentiated liposarcoma.
Genes Chromosomes Cancer
PUBLISHED: 06-17-2011
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Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31, CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) "single nucleotide polymorphism"/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at Y436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma.
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A novel bioinformatics pipeline for identification and characterization of fusion transcripts in breast cancer and normal cell lines.
Nucleic Acids Res.
PUBLISHED: 05-27-2011
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SnowShoes-FTD, developed for fusion transcript detection in paired-end mRNA-Seq data, employs multiple steps of false positive filtering to nominate fusion transcripts with near 100% confidence. Unique features include: (i) identification of multiple fusion isoforms from two gene partners; (ii) prediction of genomic rearrangements; (iii) identification of exon fusion boundaries; (iv) generation of a 5-3 fusion spanning sequence for PCR validation; and (v) prediction of the protein sequences, including frame shift and amino acid insertions. We applied SnowShoes-FTD to identify 50 fusion candidates in 22 breast cancer and 9 non-transformed cell lines. Five additional fusion candidates with two isoforms were confirmed. In all, 30 of 55 fusion candidates had in-frame protein products. No fusion transcripts were detected in non-transformed cells. Consideration of the possible functions of a subset of predicted fusion proteins suggests several potentially important functions in transformation, including a possible new mechanism for overexpression of ERBB2 in a HER-positive cell line. The source code of SnowShoes-FTD is provided in two formats: one configured to run on the Sun Grid Engine for parallelization, and the other formatted to run on a single LINUX node. Executables in PERL are available for download from our web site: http://mayoresearch.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.
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miRNA expression in colon polyps provides evidence for a multihit model of colon cancer.
PLoS ONE
PUBLISHED: 03-04-2011
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Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n?=?53) and inherited colon cancer (n?=?11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (?4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (?2-fold change).
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Changes in cardiac gene expression after pig-to-primate orthotopic xenotransplantation.
Xenotransplantation
PUBLISHED: 02-24-2011
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Gene profiling methods have been widely useful for delineating changes in gene expression as an approach for gaining insight into the mechanism of rejection or disease pathology. Herein, we use gene profiling to compare changes in gene expression associated with different orthotopic cardiac xenotransplantation (OCXTx) outcomes and to identify potential effects of OCXTx on cardiac physiology.
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Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.
PLoS ONE
PUBLISHED: 02-03-2011
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We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5 end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.
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Differential expression of microRNAs in tumors from chronically inflamed or genetic (APC(Min/+)) models of colon cancer.
PLoS ONE
PUBLISHED: 01-31-2011
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Chronic inflammation associated with ulcerative colitis predisposes individuals to increased colon cancer risk. The aim of these studies was to identify microRNAs that are aberrantly regulated during inflammation and may participate in transformation of colonic epithelial cells in the inflammatory setting.
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Drug efflux by breast cancer resistance protein is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924.
Mol. Cancer Ther.
PUBLISHED: 01-12-2011
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Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism.
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Unique cellular and mitochondrial defects mediate FK506-induced islet ?-cell dysfunction.
Transplantation
PUBLISHED: 01-05-2011
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To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506).
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Casp8p41 expression in primary T cells induces a proinflammatory response.
AIDS
PUBLISHED: 03-20-2010
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HIV infection of CD4 T cells can lead to HIV protease-mediated cleavage of procaspase 8 generating a novel, HIV-specific peptide called Casp8p41. Casp8p41 has at least two biologic functions: induction of cell death via mitochondrial depolarization and release of cytochrome C, as well as activation of nuclear factor kappa B (NFkappaB). We have previously shown that Casp8p41-induced NFkappaB activation enhances HIV LTR transcription and consequently increases HIV replication. Herein, we questioned whether Casp8p41-induced NFkappaB activation impacts the cytokine profile of cells expressing Casp8p41.
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Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform.
BMC Med Genomics
PUBLISHED: 02-25-2010
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The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.
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Gene transcription changes in asthmatic chronic rhinosinusitis with nasal polyps and comparison to those in atopic dermatitis.
PLoS ONE
PUBLISHED: 02-25-2010
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Asthmatic chronic rhinosinusitis with nasal polyps (aCRSwNP) is a common disruptive eosinophilic disease without effective medical treatment. Therefore, we sought to identify gene expression changes, particularly those occurring early, in aCRSwNP. To highlight expression changes associated with eosinophilic epithelial inflammation, we further compared the changes in aCRSwNP with those in a second eosinophilic epithelial disease, atopic dermatitis (AD), which is also closely related to asthma.
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MicroRNA networks in mouse lung organogenesis.
PLoS ONE
PUBLISHED: 02-10-2010
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MicroRNAs (miRNAs) are known to be important regulators of both organ development and tumorigenesis. MiRNA networks and their regulation of messenger RNA (mRNA) translation and protein expression in specific biological processes are poorly understood.
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Tumor transcriptome sequencing reveals allelic expression imbalances associated with copy number alterations.
PLoS ONE
PUBLISHED: 01-29-2010
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Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.
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Carboxypeptidase M: a biomarker for the discrimination of well-differentiated liposarcoma from lipoma.
Mod. Pathol.
PUBLISHED: 10-09-2009
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The discrimination between well-differentiated liposarcomas/atypical lipomatous tumors and lipomas can be diagnostically challenging at the histological level. However, cytogenetic identification of ring and giant rod chromosomes supports the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumor. These abnormal chromosomes are mainly composed of amplified genomic sequences derived from chromosome 12q13-15, and contain several genes, including MDM2, CDK4 (SAS), TSPAN31, HMGA2, and others. MDM2 is consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors, and up to 25% in other sarcomas. As part of a large genomic study of lipomatous neoplasms, we initially found CPM to be consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors. To further explore this initial finding, we investigated the copy number status of MDM2 and CPM by fluorescent in situ hybridization (FISH) on a series of 138 tumors and 17 normal tissues, including 32 well-differentiated liposarcoma/atypical lipomatous tumors, 63 lipomas, 11 pleomorphic lipomas, 2 lipoblastomas, 30 other tumors and 17 normal fat samples. All 32 well-differentiated liposarcoma/atypical lipomatous tumors showed amplification of MDM2 and CPM, usually >20 copies per cell. The other tumors lacked MDM2 and/or CPM amplification. Chromogenic in situ hybridization confirmed the above results on a subset of these tumors (n=27). These findings suggest that identification of CPM amplification could be used as an alternative diagnostic tool for the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumors.
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Role of AKT-glycogen synthase kinase axis in monocyte activation in human beings with and without type 2 diabetes.
J. Cell. Mol. Med.
PUBLISHED: 09-14-2009
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Monocyte activation by chemokines is a vital trigger for initiation of atherosclerotic process. Circulating levels of platelet activating factor (PAF), a recognized chemokine, is known to be increased in type 2 diabetes that is linked to accelerated atherosclerosis. To explore the molecular basis we examined the signalling pathways involved in PAF induced monocyte activation. PAF increased migration in monocytes obtained from THP-1 cells, nondiabetic and diabetic subjects. This effect was blocked by AKT inhibition. It did so by phosphorylation of glycogen synthase kinase (GSK)-3betaS(9), which was completely blocked by AKT inhibition. Additionally, PAF induced GSK-3beta phosphorylation was linked to Rac-1 activation and Rho-A inactivation leading to migration. Paradoxically, inhibition of GSK-3beta phosphorylation also augmented monocyte migration in THP-1, ND and diabetic monocytes through phosphorylation of AKT and activation of Rho-A that was independent of GSK. This was validated when (i) overexpression of dominant negative mutants of Rho-A reversed GSK inhibitor induced monocyte migration and (ii) AKT inhibition blocked GSK inhibitor induced Rho-A activity. Constitutively active ARAP3 (Rho-GAP) appears to have a regulatory role in monocyte activity during GSK inhibition. Finally, inhibition of monocyte GSK-3beta activity (by inhibitors and genetic manipulation) led to enhanced migration in diabetes compared to persons without diabetes. We conclude that diabetic monocytes show increased migratory capacity in response to GSK-3beta inhibition. GSK inhibitors developed to treat the metabolic complications of diabetes should therefore be used with caution.
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3 tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer.
BMC Genomics
PUBLISHED: 07-06-2009
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Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3 DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC).
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GLOSSI: a method to assess the association of genetic loci-sets with complex diseases.
BMC Bioinformatics
PUBLISHED: 04-03-2009
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The developments of high-throughput genotyping technologies, which enable the simultaneous genotyping of hundreds of thousands of single nucleotide polymorphisms (SNP) have the potential to increase the benefits of genetic epidemiology studies. Although the enhanced resolution of these platforms increases the chance of interrogating functional SNPs that are themselves causative or in linkage disequilibrium with causal SNPs, commonly used single SNP-association approaches suffer from serious multiple hypothesis testing problems and provide limited insights into combinations of loci that may contribute to complex diseases. Drawing inspiration from Gene Set Enrichment Analysis developed for gene expression data, we have developed a method, named GLOSSI (Gene-loci Set Analysis), that integrates prior biological knowledge into the statistical analysis of genotyping data to test the association of a group of SNPs (loci-set) with complex disease phenotypes. The most significant loci-sets can be used to formulate hypotheses from a functional viewpoint that can be validated experimentally.
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Early gene expression changes induced by the bacterial superantigen staphylococcal enterotoxin B and its modulation by a proteasome inhibitor.
Physiol. Genomics
PUBLISHED: 03-31-2009
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Toxic shock syndrome (TSS) is an acute, serious systemic illness caused by bacterial superantigens. Nonavailability of a suitable animal model until recently has hampered an in-depth understanding of the pathogenesis of TSS. In the current study, we characterized the early molecular events underlying TSS using our HLA-DR3 transgenic mouse model. Gene expression profiling using DNA microarrays identified a rapid and significant upregulation of several pro- as well as anti-inflammatory mediators, many of which have never been previously described in TSS. In vivo administration of staphylococcal enterotoxin B (SEB) led to an increase in the expression of Th0- (IL-2, 240-fold); Th1- (IFN-gamma, 360-fold; IL-12, 8-fold); Th2- (IL-4, 53-fold; IL-5, 4-fold) as well as Th17-type cytokines (IL-21, 19-fold; IL-17, 5-fold). The immunoregulatory cytokines (IL-6, 700-fold; IL-10, 18-fold); CC chemokines (such as CCL 2, 11, 3, 24, 17, 12, 7), CXC chemokines (such as CXCL 1, 2, 5, 11, 10, 19); and several proteases (matrix metalloproteinases 13, 8, 3, and 9) were also upregulated. Serum levels of several of these cytokines/chemokines were also significantly elevated. Pathway analyses revealed significant modulation in a variety of biochemical and cellular functions, providing molecular insights into the pathogenesis of TSS. Administration of bortezomib, a clinically approved proteasome inhibitor capable of blocking NF-kappaB pathway, was able to significantly modulate the expression of a variety of genes induced by SEB. Thus, our study showed that TSS is a complex process and emphasized the potential of use of bortezomib in the therapy of superantigen-induced TSS.
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Genome-wide linkage analysis for uric acid in families enriched for hypertension.
Nephrol. Dial. Transplant.
PUBLISHED: 03-03-2009
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Uric acid is heritable and associated with hypertension and insulin resistance. We sought to identify genomic regions influencing serum uric acid in families in which two or more siblings had hypertension.
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A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.
Science
PUBLISHED: 02-26-2009
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Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.
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Genome-wide transcriptional profiling reveals microRNA-correlated genes and biological processes in human lymphoblastoid cell lines.
PLoS ONE
PUBLISHED: 02-12-2009
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Expression level of many genes shows abundant natural variation in human populations. The variations in gene expression are believed to contribute to phenotypic differences. Emerging evidence has shown that microRNAs (miRNAs) are one of the key regulators of gene expression. However, past studies have focused on the miRNA target genes and used loss- or gain-of-function approach that may not reflect natural association between miRNA and mRNAs.
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Effect of testosterone on insulin stimulated IRS1 Ser phosphorylation in primary rat myotubes--a potential model for PCOS-related insulin resistance.
PLoS ONE
PUBLISHED: 01-26-2009
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Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1(636/639) in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.
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Chronic caloric restriction preserves mitochondrial function in senescence without increasing mitochondrial biogenesis.
Cell Metab.
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Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis.
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Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement.
Am. J. Physiol. Endocrinol. Metab.
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Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-¹³C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion (n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2-7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70(S6K), and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.
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A two-stage evaluation of genetic variation in immune and inflammation genes with risk of non-Hodgkin lymphoma identifies new susceptibility locus in 6p21.3 region.
Cancer Epidemiol. Biomarkers Prev.
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Non-Hodgkin lymphoma (NHL) is a malignancy of lymphocytes, and there is growing evidence for a role of germline genetic variation in immune genes in NHL etiology.
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Germline copy number variation and ovarian cancer survival.
Front Genet
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Copy number variants (CNVs) have been implicated in many complex diseases. We examined whether inherited CNVs were associated with overall survival among women with invasive epithelial ovarian cancer. Germline DNA from 1,056 cases (494 deceased, average of 3.7?years follow-up) was interrogated with the Illumina 610 quad genome-wide array containing, after quality control exclusions, 581,903 single nucleotide polymorphisms (SNPs) and 17,917 CNV probes. Comprehensive analysis capitalized upon the strengths of three complementary approaches to CNV classification. First, to identify small CNVs, single markers were evaluated and, where associated with survival, consecutive markers were combined. Two chromosomal regions were associated with survival using this approach (14q31.3 rs2274736 p?=?1.59?×?10(-6), p?=?0.001; 22q13.31 rs2285164 p?=?4.01?×?10(-5), p?=?0.009), but were not significant after multiple testing correction. Second, to identify large CNVs, genome-wide segmentation was conducted to characterize chromosomal gains and losses, and association with survival was evaluated by segment. Four regions were associated with survival (1q21.3 loss p?=?0.005, 5p14.1 loss p?=?0.004, 9p23 loss p?=?0.002, and 15q22.31 gain p?=?0.002); however, again, after correcting for multiple testing, no regions were statistically significant, and none were in common with the single marker approach. Finally, to evaluate associations with general amounts of copy number changes across the genome, we estimated CNV burden based on genome-wide numbers of gains and losses; no associations with survival were observed (p?>?0.40). Although CNVs that were not well-covered by the Illumina 610 quad array merit investigation, these data suggest no association between inherited CNVs and survival after ovarian cancer.
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Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas.
Blood
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Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ? 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ?Np63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.
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Search for chromosome rearrangements: new approaches toward discovery of novel translocations in head and neck squamous cell carcinoma.
Head Neck
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Chromosome rearrangements that result in gene fusions have important roles in the initial steps of tumorigenesis, especially in leukemias and lymphomas, but the biological and clinical impact of gene fusions in common solid tumors are less understood. The purpose of this study was to discover novel translocations that could result in gene fusions in oropharyngeal squamous cell carcinomas (OPSCCs).
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SAAP-RRBS: streamlined analysis and annotation pipeline for reduced representation bisulfite sequencing.
Bioinformatics
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Reduced representation bisulfite sequencing (RRBS) is a cost-effective approach for genome-wide methylation pattern profiling. Analyzing RRBS sequencing data is challenging and specialized alignment/mapping programs are needed. Although such programs have been developed, a comprehensive solution that provides researchers with good quality and analyzable data is still lacking. To address this need, we have developed a Streamlined Analysis and Annotation Pipeline for RRBS data (SAAP-RRBS) that integrates read quality assessment/clean-up, alignment, methylation data extraction, annotation, reporting and visualization. This package facilitates a rapid transition from sequencing reads to a fully annotated CpG methylation report to biological interpretation.
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Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations.
Front Oncol
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KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NF?B, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene-gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NF?B, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPAR? signaling pathways, suggesting that targeted PPAR? antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.
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MicroRNA-mRNA interactions in a murine model of hyperoxia-induced bronchopulmonary dysplasia.
BMC Genomics
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Bronchopulmonary dysplasia is a chronic lung disease of premature neonates characterized by arrested pulmonary alveolar development. There is increasing evidence that microRNAs (miRNAs) regulate translation of messenger RNAs (mRNAs) during lung organogenesis. The potential role of miRNAs in the pathogenesis of BPD is unclear.
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Early life sun exposure, vitamin D-related gene variants, and risk of non-Hodgkin lymphoma.
Cancer Causes Control
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It has been hypothesized that vitamin D mediates the inverse relationship between sun exposure and non-Hodgkin lymphoma (NHL) risk reported in several recent studies. We evaluated the association of self-reported sun exposure at ages <13, 13-21, 22-40, and 41+ years and 19 single nucleotide polymorphisms (SNPs) from 4 candidate genes relevant to vitamin D metabolism (RXR, VDR , CYP24A1, CYP27B1) with NHL risk.
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Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides.
Blood
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The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.
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Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing.
Oncotarget
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Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.
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Detection of redundant fusion transcripts as biomarkers or disease-specific therapeutic targets in breast cancer.
Cancer Res.
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Fusion genes and fusion gene products are widely employed as biomarkers and therapeutic targets in hematopoietic cancers, but their applications have yet to be appreciated in solid tumors. Here, we report the use of SnowShoes-FTD, a powerful new analytic pipeline that can identify fusion transcripts and assess their redundancy and tumor subtype-specific distribution in primary tumors. In a study of primary breast tumors, SnowShoes-FTD was used to analyze paired-end mRNA-Seq data from a panel of estrogen receptor (ER)(+), HER2(+), and triple-negative primary breast tumors, identifying tumor-specific fusion transcripts by comparison with mRNA-Seq data from nontransformed human mammary epithelial cell cultures plus the Illumina Body Map data from normal tissues. We found that every primary breast tumor that was analyzed expressed one or more fusion transcripts. Of the 131 tumor-specific fusion transcripts identified, 86 were "private" (restricted to a single tumor) and 45 were "redundant" (distributed among multiple tumors). Among the redundant fusion transcripts, 7 were unique to ER(+) tumors and 8 were unique to triple-negative tumors. In contrast, none of the redundant fusion transcripts were unique to HER2(+) tumors. Both private and redundant fusion transcripts were widely expressed in primary breast tumors, with many mapping to genomic loci implicated in breast carcinogenesis and/or risk. Our finding that some fusion transcripts are tumor subtype-specific suggests that these entities may be critical determinants in the etiology of breast cancer subtypes, useful as biomarkers for tumor stratification, or exploitable as cancer-specific therapeutic targets.
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Concordance of changes in metabolic pathways based on plasma metabolomics and skeletal muscle transcriptomics in type 1 diabetes.
Diabetes
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Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry-based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome-based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.
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Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.
Proc. Natl. Acad. Sci. U.S.A.
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To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.