Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay

Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA’s capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.National Institutes of Health (U.S.) (grant DP2OD006514)National Institutes of Health (U.S.) (grant K99HG0081)National Institutes of Health (U.S.) (grant R01HG006785

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis

Sciviewer enables interactive visual interrogation of single-cell RNA-Seq data from the Python programming environment [preprint]

Visualizing two-dimensional (2D) embeddings (e.g. UMAP or tSNE) is a key step in interrogating single-cell RNA sequencing (scRNA-Seq) data. Subsequently, users typically iterate between programmatic analyses (e.g. clustering and differential expression) and visual exploration (e.g. coloring cells by interesting features) to uncover biological signals in the data. Interactive tools exist to facilitate visual exploration of embeddings such as performing differential expression on user-selected cells. However, the practical utility of these tools is limited because they don’t support rapid movement of data and results to and from the programming environments where the bulk of data analysis takes place, interrupting the iterative process. Here, we present the Single-cell Interactive Viewer (Sciviewer), a tool that overcomes this limitation by allowing interactive visual interrogation of embeddings from within Python. Beyond differential expression analysis of user-selected cells, Sciviewer implements a novel method to identify genes varying locally along any user-specified direction on the embedding. Sciviewer enables rapid and flexible iteration between interactive and programmatic modes of scRNA-Seq exploration, illustrating a useful approach for analyzing high-dimensional data

Medical education in the United States: do residents feel prepared?

Background: Medical schools face a growing challenge in providing a comprehensive educational experience. Students must graduate with not only the medical knowledge but also the requisite skills to care for patients and serve as physicians-in-training. Objective: To assess whether residents felt prepared by their medical school training. Method We developed a questionnaire to assess resident attitudes towards various aspects of their medical school training and electronically distributed it among 107 United States training institutions. Results: A total of 2287 residents responded. Overall, a majority (53.8 %) agreed that ‘medical school prepared me well to be a resident.’ Most residents felt very well or mostly prepared in medical knowledge and clinical skills such as collecting a history (92.3 %), presenting a physical exam (86.1 %), or pathophysiology (81.6 %), but not for applied medical and psychosocial practices including end-of-life care (41.7 %), dealing with a patient death (46.3 %), and considering cost-effective care (28.7 %). Additionally, many residents reported feeling underprepared for time and fatigue management, debt, and medical-legal issues. Conclusions: Medical school graduates generally feel well prepared for residency. However, they may be less prepared to face important psychosocial, cultural and professional issues. Ultimately, a greater emphasis on skills and psychosocial experience may yield graduates who feel better prepared for today's residency challenges

Single-cell profiling of Ebola virus disease In Vivo reveals viral and host dynamics

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.We thank E. Normandin, K. Siddle, S. Reilly, S. Weingarten-Gabbay, C. Myhrvold, K. DeRuff, M. Rudy, N. Barkas, M. Babadi, C. Edwards, M. Reyes, N. Hacohen, A. Regev, and E. Hodis for helpful comments on this work. We thank S. Wolock for useful feedback and for providing scripts and processed data for investigating the human bone marrow samples. We thank D. Schwarz and New England Biolabs for generously providing SauCas9 and technical advice. We thank A. Matthews and M. Kemball for help in project management and administration. We thank S. Knemeyer and SciStories for illustrations. This work is supported by the US Food and Drug Administration (FDA) contracts HHSF223201810172C and HHSF223201610018C , National Institute of Allergy and Infectious Diseases (NIAID) U19AI110818 , and HHMI . This work was partially supported by NIAID Interagency agreement NOR15003-001-0000 . The nonhuman primate work completed at the NIAID Integrated Research Facility was supported in part by the NIAID Division of Intramural Research and NIAID Division of Clinical Research and was performed under Battelle Memorial Institute Contract (No. HHSN272200700016I ), and manuscript drafting was performed under Laulima Government Solutions, LLC . Contract (No. HHSN272201800013C ). J.L. performed this work as an employee of Battelle. J.R.K., B.D.-K., R.A., and R.S.B. are current employees of Laulima Government Solutions. D.K. was supported by award number T32GM007753 from the National Institute of General Medical Sciences (NIGMS). A.E.L. was supported by the National Science Foundation (NSF) under Grant No. DGE 1144152 . M.M. was a Gilead Fellow of the Life Sciences Research Foundation . K.G.B was supported by a K01 ( NIH-TW010853 ) and an ASTMH Shope fellowship . A.K.S. was supported by the Searle Scholars Program , the Beckman Young Investigator Program , a Sloan Fellowship in Chemistry , NIH 5U24AI118672 , and the Bill and Melinda Gates Foundation . The CyTOF facility at the trans-NIH Center for Human Immunology is supported by funding from the Intramural Research Program of the NIH . The authors are solely responsible for the content of this paper, which does not necessarily represent the official views of the US Department of Health and Human Services (HHS), the NIH, the NIGMS, the FDA, or the institutions and companies affiliated with the authors.Peer Reviewed"Article signat per 27autors/es: Dylan Kotliar, Aaron E Lin, James Logue, Travis K Hughes, Nadine M Khoury, Siddharth S Raju, Marc H Wadsworth, Han Chen, Jonathan R Kurtz, Bonnie Dighero-Kemp, Zach B Bjornson, Nilanjan Mukherjee, Brian A Sellers, Nancy Tran, Matthew R Bauer, Gordon C Adams, Ricky Adams, John L Rinn, Marta Melé, Stephen F Schaffner, Garry P Nolan, Kayla G Barnes, Lisa E Hensley, David R McIlwain, Alex K Shalek, Pardis C Sabeti, Richard S Bennett "Postprint (published version

Locally Disordered Methylation Forms the Basis of Intratumor Methylome Variation in Chronic Lymphocytic Leukemia

Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of 104 primary chronic lymphocytic leukemias (CLLs). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories.Stem Cell and Regenerative Biolog

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

© 2020 The Author(s) Single-cell profiling of circulating immune cells during Ebola virus (EBOV) infection in non-human primates resolves molecular correlates of viral tropism, characterizes replication dynamics within infected cells, and distinguishes expression changes that are mediated by viral infection from those due to cytokine signaling