ABSTRACT
Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.
Subject(s)
COVID-19 , Toll-Like Receptor 7 , Cytokines/metabolism , Down-Regulation , Humans , Leukocytes, Mononuclear/metabolism , Male , SARS-CoV-2 , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/genetics , Toll-Like Receptor 8/metabolismABSTRACT
The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.
Subject(s)
COVID-19/genetics , COVID-19/physiopathology , Exome Sequencing , Genetic Predisposition to Disease , Phenotype , Severity of Illness Index , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Germany , Humans , Italy , Male , Middle Aged , Polymorphism, Single Nucleotide , Quebec , SARS-CoV-2 , Sweden , United KingdomABSTRACT
OBJECTIVES: to describe the first wave of the COVID-19 pandemic with a focus on undetected cases and to evaluate different post-lockdown scenarios. DESIGN: the study introduces a SEIR compartmental model, taking into account the region-specific fraction of undetected cases, the effects of mobility restrictions, and the personal protective measures adopted, such as wearing a mask and washing hands frequently. SETTING AND PARTICIPANTS: the model is experimentally validated with data of all the Italian regions, some European countries, and the US. MAIN OUTCOME MEASURES: the accuracy of the model results is measured through the mean absolute percentage error (MAPE) and Lewis criteria; fitting parameters are in good agreement with previous literature. RESULTS: the epidemic curves for different countries and the amount of undetected and asymptomatic cases are estimated, which are likely to represent the main source of infections in the near future. The model is applied to the Hubei case study, which is the first place to relax mobility restrictions. Results show different possible scenarios. Mobility and the adoption of personal protective measures greatly influence the dynamics of the infection, determining either a huge and rapid secondary epidemic peak or a more delayed and manageable one. CONCLUSIONS: mathematical models can provide useful insights for healthcare decision makers to determine the best strategy in case of future outbreaks.
Subject(s)
COVID-19/epidemiology , Models, Theoretical , Pandemics , Asymptomatic Infections/epidemiology , COVID-19/diagnosis , COVID-19/prevention & control , Convalescence , Disease Susceptibility , Environmental Exposure , Europe/epidemiology , Geography, Medical , Hand Hygiene/statistics & numerical data , Humans , Italy/epidemiology , Personal Protective Equipment/statistics & numerical data , Physical Distancing , Quarantine/statistics & numerical data , United States/epidemiologyABSTRACT
We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.
Subject(s)
COVID-19 , Humans , COVID-19/genetics , Genome-Wide Association Study , Genetic Predisposition to Disease , Exome Sequencing , PhenotypeABSTRACT
Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19.
Subject(s)
COVID-19 , Cystic Fibrosis , Humans , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Alleles , Cystic Fibrosis/pathology , COVID-19/genetics , HeterozygoteABSTRACT
The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.
Subject(s)
COVID-19 , Toll-Like Receptor 3 , Autophagy/genetics , Biomarkers , COVID-19/genetics , HEK293 Cells , Humans , Hydroxychloroquine/therapeutic use , Male , Polymorphism, Single Nucleotide , SARS-CoV-2/genetics , Severity of Illness Index , Toll-Like Receptor 3/geneticsABSTRACT
Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.
Subject(s)
COVID-19 , Host-Pathogen Interactions , SARS-CoV-2 , Sialic Acids , Spike Glycoprotein, Coronavirus , COVID-19/transmission , Cryoelectron Microscopy , Genetic Variation , Humans , Nuclear Magnetic Resonance, Biomolecular , Polysaccharides/chemistry , Protein Binding , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Sialic Acids/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.
Subject(s)
COVID-19/genetics , P-Selectin/genetics , Thrombosis/genetics , COVID-19/complications , Down-Regulation , Female , Humans , Male , Middle Aged , Point Mutation , SARS-CoV-2/isolation & purification , Thrombosis/etiologyABSTRACT
Within the GEN-COVID Multicenter Study, biospecimens from more than 1000 SARS-CoV-2 positive individuals have thus far been collected in the GEN-COVID Biobank (GCB). Sample types include whole blood, plasma, serum, leukocytes, and DNA. The GCB links samples to detailed clinical data available in the GEN-COVID Patient Registry (GCPR). It includes hospitalized patients (74.25%), broken down into intubated, treated by CPAP-biPAP, treated with O2 supplementation, and without respiratory support (9.5%, 18.4%, 31.55% and 14.8, respectively); and non-hospitalized subjects (25.75%), either pauci- or asymptomatic. More than 150 clinical patient-level data fields have been collected and binarized for further statistics according to the organs/systems primarily affected by COVID-19: heart, liver, pancreas, kidney, chemosensors, innate or adaptive immunity, and clotting system. Hierarchical clustering analysis identified five main clinical categories: (1) severe multisystemic failure with either thromboembolic or pancreatic variant; (2) cytokine storm type, either severe with liver involvement or moderate; (3) moderate heart type, either with or without liver damage; (4) moderate multisystemic involvement, either with or without liver damage; (5) mild, either with or without hyposmia. GCB and GCPR are further linked to the GCGDR, which includes data from whole-exome sequencing and high-density SNP genotyping. The data are available for sharing through the Network for Italian Genomes, found within the COVID-19 dedicated section. The study objective is to systematize this comprehensive data collection and begin identifying multi-organ involvement in COVID-19, defining genetic parameters for infection susceptibility within the population, and mapping genetically COVID-19 severity and clinical complexity among patients.
Subject(s)
Biological Specimen Banks , COVID-19/genetics , Genetic Predisposition to Disease , Registries , SARS-CoV-2 , Specimen Handling , Adolescent , Adult , COVID-19/epidemiology , Female , Humans , Italy , MaleABSTRACT
Background: Recently, loss-of-function variants in TLR7 were identified in two families in which COVID-19 segregates like an X-linked recessive disorder environmentally conditioned by SARS-CoV-2. We investigated whether the two families represent the tip of the iceberg of a subset of COVID-19 male patients. Methods: This is a nested case-control study in which we compared male participants with extreme phenotype selected from the Italian GEN-COVID cohort of SARS-CoV-2-infected participants (<60 y, 79 severe cases versus 77 control cases). We applied the LASSO Logistic Regression analysis, considering only rare variants on young male subsets with extreme phenotype, picking up TLR7 as the most important susceptibility gene. Results: Overall, we found TLR7 deleterious variants in 2.1% of severely affected males and in none of the asymptomatic participants. The functional gene expression profile analysis demonstrated a reduction in TLR7-related gene expression in patients compared with controls demonstrating an impairment in type I and II IFN responses. Conclusions: Young males with TLR7 loss-of-function variants and severe COVID-19 represent a subset of male patients contributing to disease susceptibility in up to 2% of severe COVID-19. Funding: Funded by private donors for the Host Genetics Research Project, the Intesa San Paolo for 2020 charity fund, and the Host Genetics Initiative. Clinical trial number: NCT04549831.
Subject(s)
COVID-19/genetics , Polymorphism, Single Nucleotide , Toll-Like Receptor 7/genetics , Adult , COVID-19/diagnosis , COVID-19/epidemiology , Case-Control Studies , Genetic Predisposition to Disease , HEK293 Cells , Humans , Italy/epidemiology , Male , Middle Aged , SARS-CoV-2/isolation & purification , Severity of Illness IndexABSTRACT
BACKGROUND: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. METHODS: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. FINDINGS: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). INTERPRETATION: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. FUNDING: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.