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Biology subjects

Patel, R. S.

Publications and source records attributed to Patel, R. S..

18 recordsLinked to original sources

Gene regulatory networks define human airway epithelial cell types and their distinct responses to type I interferon

The human airway epithelium (HAE) is composed of diverse cell types that coordinate essential functions and host defenses. Among these defenses, interferons (IFNs) are central to antiviral programs. However, the gene regulatory networks (GRNs) governing HAE cellular identities and their IFN responses are incompletely defined. At single-cell resolution, we characterized the transcriptomes and accessible chromatin landscapes of HAE cell types, at steady state and following IFN{beta} stimulation. The resulting scRNA-seq and snATAC-seq data informed genome-scale GRN construction and inference of transcriptional circuits underlying cell identities. In response to IFN, we identified a shared transcriptional program across HAE cell types, and an expanded set of interferon-responsive genes exhibiting cell type-associated expression patterns. Cell type-associated transcription factors and chromatin accessibility contribute to distinct IFN-responsive gene expression programs. Together, these data provide a blueprint for molecular regulation of complex HAE responses and a foundation for therapeutic strategies to enhance host antiviral defense.

systems biology↗

A Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP) for Extracellular Electron Transfer

Electroactive microbes can be used as components in electrical devices to leverage their unique behavior for biotechnology, but they remain challenging to engineer because the bioelectrochemical systems (BES) used for characterization are low-throughput. To overcome this challenge, we describe the development of the Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP), which allows for samples to be arrayed and characterized in individually addressable microwells. This device reliably reports on the current generated by electroactive bacteria on the minute time scale, decreasing the time for data acquisition by several orders of magnitude compared to conventional BES. Also, this device increased the throughput of screening engineered biological components in cells, quickly identifying mutants of the membrane protein wire MtrA in Shewanella oneidensis that retain the ability to support extracellular electron transfer (EET). BiCASP is expected to enable the design of new components for bioelectronics by supporting directed evolution of electroactive proteins. The bigger pictureDevices that interface microbes and materials, known as bioelectronics, can be used to sense environmental chemicals in real time, generate energy from sugars, and synthesize chemicals. While these devices leverage the unique capabilities of living systems as components in devices, such as their ability to convert chemical information in the environment into electrical information at the cell surface, it remains challenging to engineer these cellular components and their biomolecules for new applications, largely because commercially available bioelectrochemical systems for monitoring current generated by electroactive microbes are costly and require large culture volumes, needs continuous monitoring for days to obtain stable signals, and multichannel potentiostats to monitor multiple microbes in parallel. To overcome these challenges, we created the Bioelectrochemical Crossbar Architecture Screening Platform or BiCASP that is easy to fabricate, enables parallel analysis of microbial samples in flexible arrayed formats, and yields a stable signal on the minute time scale. This device is expected to enable the application of combinatorial protein engineering methods, such as directed evolution, to proteins that control microbial current production, by allowing for fast screening of cells expressing protein mutant libraries. As a proof-of-concept, we demonstrate that this device can screen for cells that express mutants of decaheme cytochromes that retain the ability to electrically connect cells to electrodes. This device will simplify the engineering of cells and proteins that function as electrical switches as well as the diversification of bioelectronic devices for real-time sensing of chemicals in the environment. Furthermore, BiCASP is promising as a high-throughput screening (HTS) platform, enabling rapid, parallel analysis of cellular and molecular interactions of diverse biological systems through label-free electrochemical methods. Such capabilities could transform drug discovery, personalized medicine, and functional genomics, supporting systematic genetic and chemical screens even at single-cell resolution. HighlightsO_LIA high-throughput screening platform with individual addressability C_LIO_LIA device with a flexible crossbar architecture that simplifies current analysis C_LIO_LIReproducible detection of real-time cellular current on the minute time scale C_LIO_LIThe device can be used to screen a library for cells with functional protein wires C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/663982v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@c451d1org.highwire.dtl.DTLVardef@d7a50corg.highwire.dtl.DTLVardef@194e986org.highwire.dtl.DTLVardef@16f7af9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

HIV Vpr activates a nucleolar-specific ATR pathway to degrade the nucleolar stress sensor CCDC137

The lentiviral accessory protein Vpr engages an extensive network of cellular pathways to drive diverse host consequences. Of its many phenotypes, CRL4A-E3 ubiquitin ligase complex co-option, DNA damage response (DDR) engagement, and G2/M arrest are conserved and thus proposed to be functionally important. How Vpr effects these functions and whether they explain how Vpr dysregulates additional cellular pathways remain unclear. Here we leverage the ability of Vpr to deplete the nucleolar protein CCDC137 to understand how Vpr-induced DDR activation impacts nucleolar processes. We characterize CCDC137 as an indirect Vpr target whose degradation does not correlate with Vpr-induced G2/M arrest. Yet, degradation is conserved among Vpr from the pandemic HIV-1 and related SIVcpz/SIVgor, and it is triggered by genomic insults that activate a nucleolar ATR pathway in a manner similar to camptothecin. We determine that Vpr causes ATR-dependent features of nucleolar stress that correlate with CCDC137 degradation, including redistribution of nucleolar proteins, altered nucleolar morphology, and repressed ribosome biogenesis. Together, this data distinguishes CCDC137 as a non-canonical Vpr target that may serve as a sensor of nucleolar disruption, and in doing so, identifies a novel role for Vpr in nucleolar stress. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/612530v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d43859org.highwire.dtl.DTLVardef@bf95f4org.highwire.dtl.DTLVardef@dec82org.highwire.dtl.DTLVardef@22802c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Broad-spectrum RNA antiviral inspired by ISG15-/- deficiency

Type I interferons (IFN-I) are cytokines with potent antiviral and inflammatory capacities. IFN-I signaling drives the expression of hundreds of IFN-I stimulated genes (ISGs), whose aggregate function results in the control of viral infection. A few of these ISGs are tasked with negatively regulating the IFN-I response to prevent overt inflammation. ISG15 is a negative regulator whose absence leads to persistent, low-grade elevation of ISG expression and concurrent, self-resolving mild autoinflammation. The limited breadth and low-grade persistence of ISGs expressed in ISG15 deficiency are sufficient to confer broad-spectrum antiviral resistance. Inspired by ISG15 deficiency, we have identified a nominal collection of 10 ISGs that recapitulate the broad antiviral potential of the IFN-I system. The expression of the 10 ISG collection in an IFN-I non-responsive cell line increased cellular resistance to Zika, Vesicular Stomatitis, Influenza A (IAV), and SARS-CoV-2 viruses. A deliverable prophylactic formulation of this syndicate of 10 ISGs significantly inhibited IAV PR8 replication in vivo in mice and protected hamsters against a lethal SARS-CoV-2 challenge, suggesting its potential as a broad-spectrum antiviral against many current and future emerging viral pathogens. One-Sentence SummaryHuman inborn error of immunity-guided discovery and development of a broad-spectrum RNA antiviral therapy

immunology↗

Identification of MYC synthetic lethal genes and networks

MYC is a potent oncogene that is frequently overexpressed in human tumors arising in different tissues. To date there are no approved therapies to directly antagonize oncogenic MYC and its role in driving tumorigenesis. As an alternative approach we employed genetic screens using CRISPR and shRNA to identify the genes that are required for the survival and growth of cells harboring high levels of MYC expression. We find that cells with elevated MYC require the expression of many pro-growth and metabolic pathways including genes involved in mitochondrial citrate production and transport. This citrate producing pathway is critical for cells with elevated MYC to generate the necessary acetyl-CoA to drive the lipid synthesis required for increased proliferation. Inhibition of this pathway results in reduced proliferation and in vivo tumor growth providing a potential therapeutic strategy to target MYC-driven cancers. HIGHLIGHTS- CRISPR and shRNA screens identify synthetic lethal interactions with overexpressed MYC - MYC overexpressing cells are more sensitive to disruption of citrate production and transport - Inhibition of SLC25A1 reduces growth of MYC driven tumors

cancer biology↗

A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease1-7. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE5. However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

cell biology↗

Machine-Learning Based Optimisation of a Biomimiced Herringbone Microstructure for Superior Aerodynamic Performance.

Biomimicry involves taking inspiration from existing designs in nature to generate new and efficient systems. The feathers of birds which form a characteristic herringbone riblet shape are known to effectively reduce drag. This paper aims to optimise the individual constituent structure of a herringbone riblet pattern using a combination of computational fluid dynamics (CFD) and supervised machine learning algorithms to achieve the best possible reduction in drag. Initially, a herringbone riblet design is made by computer aided designing and is parameterised. By randomly varying these parameters, 107 additional designs are made and are subjected to CFD calculations to derive their drag coefficients (Cd). These designs are used to train a supervised learning model which is employed as an alternative to CFD for predicting the Cd of other 10000 randomly generated herringbone riblet designs. Amongst these, the design with the least predicted Cd is considered as the optimised design. The Cd prediction for the optimised design had an error of 4 % with respect to its true Cd which was calculated by using CFD. The optimised design of this microstructure can be utilised for drag reduction of aeronautical, automotive or oceanic crafts by integrating them onto their surfaces.

bioengineering↗

Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor

Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.

microbiology↗

The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice

In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted (hAd5[E1-, E2b-, E3-]) that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell-surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity - including mucosal immunity - against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.

immunology↗

A recombinant ACE2 Triple Decoy that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants

The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2 decoy that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinity in vitro. A double mutant ACE2(T27Y/H34A)-IgG1FC fusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FC Triple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate delivery in vivo. Summary sentenceAn ACE2(N27Y/H34A/H374N)-IgG1FC fusion protein decoy sustains high affinity to all SARS-CoV-2 spike receptor binding domain (RBD) protein variants tested, shows enhanced affinity for the N501Y and L452R variants, and the highest affinity for combined N501Y and E484K variants.

bioengineering↗

Multi-omic Characterization of Human Tubular Epithelial Cell Response to Serum

Proteinuria, the spillage of serum proteins into the urine, is a feature of glomerulonephritides, podocyte disorders and diabetic nephropathy. However, the response of tubular epithelial cells to serum protein exposure has not been systematically characterized. Using transcriptomic profiling we studied serum-induced changes in primary human tubular epithelial cells cultured in 3D microphysiological devices. Serum proteins induced cellular proliferation, cytokine secretion and activated a coordinated stress response. We orthogonally confirmed our findings by comparing the transcriptomic and epigenomic landscapes of intact human kidney cortex and isolated tubular epithelial cells cultured in fetal bovine serum. Importantly, key transcriptomic programs in response to either type of serum exposure remained consistent, including comparisons to an established mouse model of kidney injury. This serum-induced transcriptional response was dominated by switching off of nuclear receptor-driven programs and activation of AP-1 and NF-{kappa}B signatures in the tubular epigenomic landscape. These features of active regulation were seen at canonical kidney injury genes (HAVCR1) and genes associated with COVID-19 (ACE2, IL6). Our data provide a reference map for dissecting the regulatory and transcriptional response of kidney tubular epithelial cells injury induced by serum.

cell biology↗

A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses

In response to the health crisis presented by the COVID-19 pandemic, rapid development of safe and effective vaccines that elicit durable immune responses is imperative. Recent reports have raised the concern that antibodies in COVID-19 convalescent patients may not be long lasting and thus even these individuals may require vaccination. Vaccine candidates currently in clinical testing have focused on the SARS-CoV-2 wild type spike (S) protein (S-WT) as the major antigen of choice and while pre-clinical and early clinical testing have shown that S elicits an antibody response, we believe the optimal vaccine candidate should be capable of inducing robust, durable T-cell responses as well as humoral responses. We report here on a next generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, comprising both an S sequence optimized for cell surface expression (S-Fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal subcellular compartment, with the potential to generate durable immune protection. Our studies suggest that this bivalent vaccine is optimized for immunogenicity as evidenced by the following findings: (i) The optimized S-Fusion displayed improved S receptor binding domain (RBD) cell surface expression compared to S-WT where little surface expression was detected; (ii) the expressed RBD from S-Fusion retained conformational integrity and recognition by ACE2-Fc; (iii) the viral N protein modified with an enhanced T-cell stimulation domain (ETSD) localized to endosomal/lysosomal subcellular compartments for MHC I/II presentation; and (iv) these optimizations to S and N (S-Fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T-cell responses in antigen-naive pre-clinical models. Both the T-cell and antibody immune responses to S and N demonstrated a T-helper 1 (Th1) bias. The antibody responses were neutralizing as demonstrated by two independent SARS-CoV-2 neutralization assays. Based on these findings, we are advancing this next generation bivalent hAd5 S-Fusion + N-ETSD vaccine as our lead clinical candidate to test for its ability to provide robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection. Further studies are ongoing to explore utilizing this vaccine construct in oral, intranasal, and sublingual formulations to induce mucosal immunity in addition to cell-mediated and humoral immunity. The ultimate goal of an ideal COVID-19 vaccine is to generate long-term T and B cell memory.

immunology↗

Single cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse immune cell subtypes including T-bet+ B cells

Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary (e.g. for host-pathogen interaction studies), but presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. Here, we demonstrate the utility of single cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMCs) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: Monocytes/Dendritic Cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1- lymphocytes, and Basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Unexpectedly, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells; an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms, and form the basis for an immune cell atlas of horse peripheral blood.

immunology↗

Drosophila Sex Peptide Controls the Assembly of Lipid Microcarriers in Seminal Fluid

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behaviour. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterised protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term post-mating responses including ovulation, elevated feeding and reduced receptivity to remating, primarily signalling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating, where they rapidly disassemble. Remarkably, SP is a key assembly factor for microcarriers and is also required for the female disassembly process to occur normally. Males expressing non-functional SP mutant proteins that affect SPs binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread defects in ejaculate function. Our data therefore reveal a novel role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species, which lack the signalling functions seen in D. melanogaster. Significance StatementSeminal fluid plays a critical role in reprogramming female physiology and behaviour to promote male reproductive success. We show in the fruit fly that specific seminal proteins, including the archetypal female-reprogramming molecule Sex Peptide, are stored in male seminal secretions in association with large neutral lipid-containing microcarriers, which rapidly disperse in females. Related structures are also observed in other Sex Peptide-expressing Drosophila species. Males lacking Sex Peptide have structurally defective microcarriers, leading to abnormal cargo loading and transfer to females. Our data reveal that this key signalling molecule in Drosophila seminal fluid is also a microcarrier assembly factor that controls transfer of other seminal factors, and that this may be a more evolutionarily ancient role of this protein.

cell biology↗

Genetic drug target validation using Mendelian randomization

Mendelian randomisation analysis has emerged as an important tool to elucidate the causal relevance of a range of environmental and biological risk factors for human disease. However, inference on cause is undermined if the genetic variants used to instrument a risk factor of interest also associate with other traits that open alternative pathways to the disease (horizontal pleiotropy). We show how the no horizontal pleiotropy assumption in MR analysis is strengthened when proteins are the risk factors of interest. Proteins are the proximal effectors of biological processes encoded in the genome, and are becoming assayable on an-omics scale. Moreover, proteins are the targets of most medicines, so Mendelian randomization (MR) studies of drug targets are becoming a fundamental tool in drug development. To enable such studies we introduce a formal mathematical framework that contrasts MR analysis of proteins with that of risk factors located more distally in the causal chain from gene to disease. Finally, we illustrate key model decisions and introduce an analytical framework for maximizing power and elucidating the robustness of drug target MR analyses.

genetics↗

Bleeding in cardiac patients prescribed antithrombotic drugs: Electronic health record phenotyping algorithms, incidence, trends and prognosis

BackgroundClinical guidelines and public health authorities lack recommendations on scalable approaches to defining and monitoring the occurrence and severity of bleeding in populations prescribed antithrombotic therapy. We aimed to develop electronic health record algorithms for different bleeding phenotypes, and to determine the incidence, time trends and prognosis of bleeding in patients with incident cardiac disorders indicated for antiplatelet and/or vitamin K antagonist (VKA) therapy. MethodsWe examined linked primary care, hospital admission and death registry electronic health records (CALIBER 1998-2010, England) of patients with newly diagnosed atrial fibrillation, acute myocardial infarction, unstable angina or stable angina to develop algorithms for bleeding events. Kaplan-Meier plots were used to estimate the incidence of bleeding events and we used Cox regression models to assess prognosis for all-cause mortality, atherothrombotic events and further bleeding following bleeding events. ResultsWe present electronic health record phenotyping algorithms for bleeding based on bleeding diagnosis in primary or hospital care, symptoms, transfusion, surgical procedures, and haemoglobin values. In validation of the phenotype we estimated a positive predictive value of 0.88 (95% Cl: 0.64, 0.99) for hospitalised bleeding. Amongst 128,815 patients, 27259 (21.2%) had at least one bleeding event, with 5 year risks of bleeding of 29.1%, 21.9%, 25.3% and 23.4% following diagnoses of atrial fibrillation, acute myocardial infarction, unstable angina and stable angina respectively. Rates of hospitalised bleeding per 1000 patients more than doubled from 1.02 (95% Cl: 0.83, 1.22) in January 1998 to 2.68 (95% Cl: 2.49, 2.88) in December 2009 coinciding with increased rates of antiplatelet and VKA prescribing. Patients with hospitalised bleeding and primary care bleeding, with or without markers of severity, were at increased risk of all-cause mortality and atherothrombotic events compared to those with no bleeding. For example the hazard ratio for all-cause mortality was 1.98 (95% Cl: 1.86, 2.11) for primary care bleeding with markers of severity, and 1.99 (95% Cl: 1.92, 2.05) for hospitalised bleeding without markers of severity, compared to patients with no bleeding. ConclusionsElectronic health record bleeding phenotyping algorithms offer a scalable approach to monitoring bleeding in the population. Incidence of bleeding has doubled in incidence since 1998, affects 1 in 4 cardiac patients, and is associated with poor prognosis. Efforts are required to tackle this iatrogenic epidemic. O_LSTWhat is already known?C_LSTO_LIClinical guidelines and public health authorities lack recommendations on how to define or monitor the occurrence and severity of bleeding in populations. C_LIO_LIThis is particularly important because clinical guidelines increasingly recommend the use of one, two or three antiplatelet and vitamin K antagonist drugs to lower the risk of subsequent atherothrombotic events in common heart diseases including atrial fibrillation, acute coronary syndromes and chronic stable angina. C_LIO_LIClinical guidelines lack consistent recommendations of how to reduce the main side effect of bleeding. C_LIO_LIFor acute myocardial infarction it has been shown that combining primary care electronic health records (which include information from hospital discharge summaries) and hospital admission data can generate valid EHR disease phenotypes and provide real-world estimates of disease occurrence. C_LI O_LSTWhat is not known?C_LSTO_LIIt is not known how to define bleeding occurrence and severity in large scale, unselected populations by combining available information on bleeding diagnosis in primary or hospital care, symptoms, transfusion, surgical procedures, and haemoglobin values. C_LIO_LIThe population-based incidence, time trends and long-term prognosis of bleeding have not been evaluated in people with common cardiac disorders. C_LIO_LIComparisons of the population burden of bleeding across common cardiac disorders, such as atrial fibrillation, acute coronary syndromes and stable angina, are lacking. C_LI O_LSTWhat this study adds?C_LSTO_LIPhenotype: We developed standardised replicable EHR phenotyping algorithms defining bleeding and severity measures based on available clinical information across structured primary and hospital care EHR sources. C_LIO_LIIncidence: At 5 years of follow-up, one in five patients with cardiac disease had a bleeding event and 6.5% had fatal or severe bleeding. C_LIO_LITrends: There was approximately a two-fold increase in incidence of primary care and hospitalised bleeding between 1998 and 2010. The rate of fatal bleeding remained stable. C_LIO_LIPrognosis: Patients with bleeding recorded in primary care or in hospital admissions are at increased bleeding between 1998 and 2010. The rate of fatal bleeding remained stable, risk of all-cause death and atherothrombotic events. C_LI

epidemiology↗

Adjustment for index event bias in genome-wide association studies of subsequent events

Following numerous genome-wide association studies of disease susceptibility, there is increasing interest in genetic associations with disease prognosis, survival or other subsequent events. Such associations are vulnerable to index event bias, by which selection of subjects according to their disease status creates biased associations if common causes of incidence and prognosis are not accounted for. We propose a novel adjustment for index event bias using the residuals from the regression of genetic effects on prognosis on genetic effects on incidence. Our approach eliminates this bias when direct genetic effects on incidence and prognosis are independent, and otherwise reduces bias in realistic situations. In a study of idiopathic pulmonary fibrosis, we resolved a paradoxical association of the strong susceptibility gene MUC5B with increased survival, identifying instead a significant association with decreased survival. In re-analysis of a study of Crohns disease prognosis, four regions remained associated at genome-wide significance albeit with increased P-values.

epidemiology↗

Genomic risk prediction of coronary artery disease in nearly 500,000 adults: implications for early screening and primary prevention

BackgroundCoronary artery disease (CAD) has substantial heritability and a polygenic architecture; however, genomic risk scores have not yet leveraged the totality of genetic information available nor been externally tested at population-scale to show potential utility in primary prevention.\n\nMethodsUsing a meta-analytic approach to combine large-scale genome-wide and targeted genetic association data, we developed a new genomic risk score for CAD (metaGRS), consisting of 1.7 million genetic variants. We externally tested metaGRS, individually and in combination with available conventional risk factors, in 22,242 CAD cases and 460,387 non-cases from UK Biobank.\n\nFindingsIn UK Biobank, a standard deviation increase in metaGRS had a hazard ratio (HR) of 1.71 (95% CI 1.68-1.73) for CAD, greater than any other externally tested genetic risk score. Individuals in the top 20% of the metaGRS distribution had a HR of 4.17 (95% CI 3.97-4.38) compared with those in the bottom 20%. The metaGRS had higher C-index (C=0.623, 95% CI 0.615-0.631) for incident CAD than any of four conventional factors (smoking, diabetes, hypertension, and body mass index), and addition of the metaGRS to a model of conventional risk factors increased C-index by 3.7%. In individuals on lipid-lowering or anti-hypertensive medications at recruitment, metaGRS hazard for incident CAD was significantly but only partially attenuated with HR of 2.83 (95% CI 2.61- 3.07) between the top and bottom 20% of the metaGRS distribution.\n\nInterpretationRecent genetic association studies have yielded enough information to meaningfully stratify individuals using the metaGRS for CAD risk in both early and later life, thus enabling targeted primary intervention in combination with conventional risk factors. The metaGRS effect was partially attenuated by lipid and blood pressure-lowering medication, however other prevention strategies will be required to fully benefit from earlier genomic risk stratification.\n\nFundingNational Health and Medical Research Council of Australia, British Heart Foundation, Australian Heart Foundation.

genetics↗