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1.
Nature ; 602(7896): 268-273, 2022 02.
Article in English | MEDLINE | ID: covidwho-1671587

ABSTRACT

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.


Subject(s)
Autism Spectrum Disorder , Genetic Predisposition to Disease , Neurons , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/pathology , Cerebral Cortex/cytology , DNA-Binding Proteins/genetics , GABAergic Neurons/metabolism , GABAergic Neurons/pathology , Histone-Lysine N-Methyltransferase/genetics , Humans , Neurons/classification , Neurons/metabolism , Neurons/pathology , Organoids/cytology , Proteomics , RNA-Seq , Single-Cell Analysis , Transcription Factors/genetics
2.
Eur J Clin Invest ; 51(12): e13626, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1273086

ABSTRACT

BACKGROUND: Fever-7 is a test evaluating host mRNA expression levels of IFI27, JUP, LAX, HK3, TNIP1, GPAA1 and CTSB in blood able to detect viral infections. This test has been validated mostly in hospital settings. Here we have evaluated Fever-7 to identify the presence of respiratory viral infections in a Community Health Center. METHODS: A prospective study was conducted in the "Servicio de Urgencias de Atención Primaria" in Salamanca, Spain. Patients with clinical signs of respiratory infection and at least one point in the National Early Warning Score were recruited. Fever-7 mRNAs were profiled on a Nanostring nCounter® SPRINT instrument from blood collected upon patient enrolment. Viral diagnosis was performed on nasopharyngeal aspirates (NPAs) using the Biofire-RP2 panel. RESULTS: A respiratory virus was detected in the NPAs of 66 of the 100 patients enrolled. Median National Early Warning Score was 7 in the group with no virus detected and 6.5 in the group with a respiratory viral infection (P > .05). The Fever-7 score yielded an overall AUC of 0.81 to predict a positive viral syndromic test. The optimal operating point for the Fever-7 score yielded a sensitivity of 82% with a specificity of 71%. Multivariate analysis showed that Fever-7 was a robust marker of viral infection independently of age, sex, major comorbidities and disease severity at presentation (OR [CI95%], 3.73 [2.14-6.51], P < .001). CONCLUSIONS: Fever-7 is a promising host immune mRNA signature for the early identification of a respiratory viral infection in the community.


Subject(s)
RNA, Messenger/blood , Respiratory Tract Infections/diagnosis , Virus Diseases/diagnosis , Adaptor Proteins, Vesicular Transport/genetics , Aged , Aged, 80 and over , Cathepsin B/genetics , DNA-Binding Proteins/genetics , Early Warning Score , Female , Gene Expression Profiling , Humans , Male , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Nasopharynx/virology , Respiratory Tract Infections/blood , Respiratory Tract Infections/genetics , Transcriptome , Virus Diseases/blood , Virus Diseases/genetics , gamma Catenin/genetics
3.
Nat Commun ; 12(1): 2735, 2021 05 12.
Article in English | MEDLINE | ID: covidwho-1241460

ABSTRACT

Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.


Subject(s)
CARD Signaling Adaptor Proteins/metabolism , DNA-Binding Proteins/metabolism , Immunity, Innate/physiology , Inflammasomes/metabolism , CARD Signaling Adaptor Proteins/genetics , Cryoelectron Microscopy , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation/genetics , Protein Structure, Secondary , Signal Transduction/physiology
4.
J Zoo Wildl Med ; 51(4): 733-744, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1041161

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (Panthera tigris altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids.


Subject(s)
COVID-19/veterinary , Feces/virology , Lions/virology , SARS-CoV-2 , Tigers/virology , Animals , Animals, Zoo , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/transmission , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , New York City/epidemiology , Transcription Factors/genetics , Transcription Factors/isolation & purification
5.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: covidwho-982503

ABSTRACT

Animal models recapitulating human COVID-19 disease, especially severe disease, are urgently needed to understand pathogenesis and to evaluate candidate vaccines and therapeutics. Here, we develop novel severe-disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than those in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and was uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2 but also play an early role in protection from acute disease.IMPORTANCE Syrian hamsters are in use as a model of disease caused by SARS-CoV-2. Pathology is pronounced in the upper and lower respiratory tract, and disease signs and endpoints include weight loss and viral RNA and/or infectious virus in swabs and organs (e.g., lungs). However, a high dose of virus is needed to produce disease, and the disease resolves rapidly. Here, we demonstrate that immunosuppressed hamsters are susceptible to low doses of virus and develop more severe and prolonged disease. We demonstrate the efficacy of a novel neutralizing monoclonal antibody using the cyclophosphamide transient suppression model. Furthermore, we demonstrate that RAG2 knockout hamsters develop severe/fatal disease when exposed to SARS-CoV-2. These immunosuppressed hamster models provide researchers with new tools for evaluating therapies and vaccines and understanding COVID-19 pathogenesis.


Subject(s)
Coronavirus Infections/immunology , Coronavirus Infections/pathology , Disease Models, Animal , Mesocricetus , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Adaptive Immunity , Animals , Animals, Genetically Modified , Betacoronavirus/physiology , COVID-19 , Cyclophosphamide , DNA-Binding Proteins/genetics , Gene Knockout Techniques , Immunosuppressive Agents , Pandemics , SARS-CoV-2 , Severity of Illness Index
6.
Eur J Med Genet ; 63(12): 104098, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-917288

ABSTRACT

INTRODUCTION: Mainstreamed genetic testing (MGT) obviates the need for a cancer genetics consultation, since trained oncologists (O) and gynaecologists (G) provide counseling, prescribe testing and deliver results. We report results from our MGT program and emphasize its utility during the COVID-19 lockdown, when cancer genetics clinics had suspended their activity. METHODS: An MGT pathway for breast and ovarian cancer (BC/OC) patients was established in Jan-2018 between the Assistance Publique - Hôpitaux de Paris.Sorbonne Université Cancer Genetics team and the Oncology/Gynecology departments at one teaching and two regional hospitals. Trained O + G evaluated patients with the Manchester Scoring System. A 12-point threshold was recommended for testing. Next-generation sequencing of BRCA1, BRCA2, PALB2, RAD51C and RAD51D was performed. Results were delivered to the patient by O/G. Pathogenic variants (PV) carriers were referred to the genetics clinic. Results are reported for the 2nd-Jan-2018 to 1st-June-2020 period. That includes the eight-week COVID-19 lockdown and three-week de-confinement phase 1. RESULTS: Results were available for 231/234 patients. Twenty-eight (12.1%) carried a PV. Of the 27 patients tested during the COVID-19 period, three carried a PV, two in BRCA1 and one in RAD51C. The clinical impact was immediate for the two BRCA1 BC cases undergoing neo-adjuvant chemotherapy, since double mastectomy and salpingo-oophorectomy will now be performed using two-step strategies. CONCLUSIONS: MGT guaranteed care continuity in BC/OC patients during the critical phases of the COVID-19 pandemic, with immediate implications for PV carriers. More broadly, we report for the first time the successful implementation of MGT in France.


Subject(s)
Breast Neoplasms/genetics , COVID-19/epidemiology , Genetic Testing , Hereditary Breast and Ovarian Cancer Syndrome/genetics , Ovarian Neoplasms/genetics , Pandemics , Adult , Aged , Aged, 80 and over , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/surgery , DNA-Binding Proteins/genetics , Fanconi Anemia Complementation Group N Protein/genetics , Female , Genetic Counseling , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Humans , Male , Mastectomy , Middle Aged , Neoadjuvant Therapy , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/surgery , Paris/epidemiology , Salpingo-oophorectomy , Young Adult
8.
JAMA Cardiol ; 5(10): 1170-1175, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-636079

ABSTRACT

Importance: Cytokine release syndrome is a complication of coronavirus disease 2019. Clinically, advanced age and cardiovascular comorbidities are the most important risk factors. Objective: To determine whether clonal hematopoiesis of indeterminate potential (CHIP), an age-associated condition with excess cardiovascular risk defined as the presence of an expanded, mutated somatic blood cell clone in persons without other hematological abnormalities, may be associated with an inflammatory gene expression sensitizing monocytes to aggravated immune responses. Design, Setting, and Participants: This hypothesis-generating diagnostic study examined a cohort of patients with severe degenerative aortic valve stenosis or chronic postinfarction heart failure, as well as age-matched healthy control participants. Single-cell RNA sequencing and analyses of circulating peripheral monocytes was done between 2017 and 2019 to assess the transcriptome of circulating monocytes. Exposures: Severe degenerative aortic valve stenosis or chronic postinfarction heart failure. Main Outcomes and Measures: CHIP-driver sequence variations in monocytes with a proinflammatory signature of genes involved in cytokine release syndrome. Results: The study included 8 patients with severe degenerative aortic valve stenosis, 6 with chronic postinfarction heart failure, and 3 healthy control participants. Their mean age was 75.7 (range, 54-89) years, and 6 were women. Mean CHIP-driver gene variant allele frequency was 4.2% (range, 2.5%-6.9%) for DNMT3A and 14.3% (range, 2.6%-37.4%) for TET2. Participants with DNMT3A or TET2 CHIP-driver sequence variations displayed increased expression of interleukin 1ß (no CHIP-driver sequence variations, 1.6217 normalized Unique Molecular Identifiers [nUMI]; DNMT3A, 5.3956 nUMI; P < .001; TET2, 10.8216 nUMI; P < .001), the interleukin 6 receptor (no CHIP-driver sequence variations, 0.5386 nUMI; DNMT3A, 0.9162 nUMI; P < .001;TET2, 0.5738 nUMI; P < .001), as well as the NLRP3 inflammasome complex (no CHIP-driver sequence variations, 0.4797 nUMI; DNMT3A, 0.9961 nUMI; P < .001; TET2, 1.2189 nUMI; P < .001), plus upregulation of CD163 (no CHIP-driver sequence variations, 0.5239 nUMI; DNMT3A, 1.4722 nUMI; P < .001; TET2, 1.0684 nUMI; P < .001), a cellular receptor capable of mediating infection, macrophage activation syndrome, and other genes involved in cytokine response syndrome. Gene ontology term analyses of regulated genes revealed that the most significantly upregulated genes encode for leukocyte-activation and interleukin-signaling pathways in monocytes of individuals with DNMT3A (myeloid leukocyte activation: log[Q value], -50.1986; log P value, -54.5177; regulation of cytokine production: log[Q value], -21.0264; log P value, -24.1993; signaling by interleukins: log[Q value], -18.0710: log P value, -21.1597) or TET2 CHIP-driver sequence variations (immune response: log[Q value], -36.3673; log P value, -40.6864; regulation of cytokine production: log[Q value], -13.1733; log P value, -16.3463; signaling by interleukins: log[Q value], -12.6547: log P value, -15.7977). Conclusions and Relevance: Monocytes of individuals who carry CHIP-driver sequence variations and have cardiovascular disease appear to be primed for excessive inflammatory responses. Further studies are warranted to address potential adverse outcomes of coronavirus disease 2019 in patients with CHIP-driver sequence variations.


Subject(s)
Aortic Valve Stenosis/complications , Clonal Hematopoiesis/genetics , Gene Expression , Heart Failure/complications , Aged , Aged, 80 and over , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , COVID-19/complications , Case-Control Studies , Cytokine Release Syndrome/genetics , Cytokines/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA-Binding Proteins/genetics , Dioxygenases , Female , Genetic Predisposition to Disease , Genetic Variation , Heterozygote , Humans , Male , Middle Aged , Monocytes , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Proto-Oncogene Proteins/genetics , Receptors, Cell Surface/metabolism , Receptors, Interleukin-6/metabolism , Transcriptome
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