Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.
Long INterspersed Element class 1 (LINE‐1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition‐competent LINE‐1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE‐1 repressors and when mutated cause the autoinflammatory disorder Aicardi‐Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE‐1 retrotransposition. It has therefore been suggested that increased LINE‐1 activity may be the cause of aberrant innate immune activation in AGS. Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE‐1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE‐1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE‐1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE‐1‐derived nucleic acids driving autoinflammation in AGS.
Transposable elements (TEs) have been considered traditionally as junk DNA, i.e., DNA sequences that despite representing a high proportion of genomes had no evident cellular functions. However, over the last decades, it has become undeniable that not only TE-derived DNA sequences have (and had) a fundamental role during genome evolution, but also TEs have important implications in the origin and evolution of many genomic disorders. This concise review provides a brief overview of the different types of TEs that can be found in genomes, as well as a list of techniques and methods used to study their impact and mobilization. Some of these techniques will be covered in detail in this Method Book.
Background OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study. Methods Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays. Results The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue. Conclusion The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.
This Article contains an error in the author affiliations. The correct affiliation for author Ruchi Shukla is ‘MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK’, and is not ‘Mater Research Institute – University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia’.
The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (Onco-MasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. Methods: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. Results: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes Z 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. Discussion: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.
Table of contents Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis -acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef prote...
Background Multigene prognostic signatures (MGPS) enable identification of candidate patients for treatment de-escalation in early stage breast cancer (BC). Here we present OncoMasTR, a MGPS for classifying the risk of distant metastasis (DM) in ER-positive, HER2-negative BC patients with up to 3 involved lymph nodes (LNs). OncoMasTR was discovered via a novel transcriptional network analysis methodology that identified genes that regulate previously identified prognostic biomarkers. These upstream genes, termed master transcriptional regulators (MTRs), were shown to provide improved prognostic performance compared with downstream genes. OncoMasTR has been mechanistically verified by RT-qPCR, immunohistochemistry and chromatin immunoprecipitation. OncoMasTR has been further trained to include clinicopathological information (CPI) to maximise its prognostic performance. Methods Two independent sample sets: 225 patients from Malmö University Hospital and 100 patients from Skåne University Hospital were used for training, cross-validation and refinement of OncoMasTR. RNA extracted from 225 archived tissues was analysed by RT-qPCR to measure the expression levels of the MTRs. Statistical models of all possible combinations of MTRs were trained and cross-validated (1,000 times x 2-fold) using the first set of 225 samples. Statistical models with the best cross-validated performance were further evaluated on RT-qPCR data from the second independent set of 100 samples. Robustness of the data was verified by assessing the reproducibility of OncoMasTR across 6 days, using 6 unique kit lots, conducted by 4 operators on 3 RT-qPCR instruments. Results In the first training set of 225 patients, OncoMasTR classified up to 72% of LN0 patients and 58% of LN0-3 patients as low risk, with ≤ 5.0% DM within each group. When incorporating CPI, its prognostic performance further improved to a c (concordance) index > 0.8. Results showed that the OncoMasTR molecular score and CPI add statistically significant prognostic value to each other. In the independent verification set, all patients with DM were correctly classified as high risk (p<0.01). In relation to reproducibility, the OncoMasTR test displayed robust performance; the molecular score coefficient of variation was 2.6% across days, kit lots, operators and instruments. Individual MTR assays demonstrated linearity over >2000-fold RNA input range and PCR efficiencies ranged from 92% to 101%. Conclusions OncoMasTR development and verification results show analytical robustness and clinically accurate risk stratification. Furthermore, OncoMasTR's binary classification of risk avoids an ambiguous intermediate risk classification and has potential to provide clinicians with useful, actionable information to support treatment decisions. The OncoMasTR test is now ready for large-scale clinical validation. Citation Format: Barron S, Jirström K, Jernström H, Ingvar C, Moran B, Wang C-JA, Loughman T, Fender B, Dynoodt P, Lopez-Ruiz C, Russell N, Gallagher WM. Prognostic value of OncoMasTR: A novel multigene signature based on master transcriptional regulators [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-06.
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