Genome-wide Association Study of Long COVID (preprint)

Infections can lead to persistent or long-term symptoms and diseases such as shingles after varicella zoster, cancers after human papillomavirus, or rheumatic fever after streptococcal infections(1,2). Similarly, infection by SARS-CoV-2 can result in Long COVID, a condition characterized by symptoms of fatigue and pulmonary and cognitive dysfunction(3-5). The biological mechanisms that contribute to the development of Long COVID remain to be clarified. We leveraged the COVID-19 Host Genetics Initiative(6,7) to perform a genome-wide association study for Long COVID including up to 6,450 Long COVID cases and 1,093,995 population controls from 24 studies across 16 countries. We identified the first genome-wide significant association for Long COVID at the FOXP4 locus. FOXP4 has been previously associated with COVID-19 severity(6), lung function(8), and cancers(9), suggesting a broader role for lung function in the pathophysiology of Long COVID. While we identify COVID-19 severity as a causal risk factor for Long COVID, the impact of the genetic risk factor located in the FOXP4 locus could not be solely explained by its association to severe COVID-19. Our findings further support the role of pulmonary dysfunction and COVID-19 severity in the development of Long COVID.

Host genetics and COVID-19 severity: increasing the accuracy of latest severity scores by Boolean quantum features (preprint)

The impact of common and rare variants in COVID-19 host genetics is widely studied in [16]. Here, common and rare variants were used to define an interpretable machine learning model for predicting COVID-19 severity. Firstly, variants were converted into 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. After that, the Boolean features, selected by these logistic models, were combined into an Integrated PolyGenic Score, the so called IPGS, which offers a very simple description of the contribution of host genetics in COVID-19 severity. IPGS leads to an accuracy of 55-60% on different cohorts and, after a logistic regression with in input both IPGS and the age, it leads to an accuracy of 75%. The goal of this paper is to improve the previous results, using the information on the host organs involved in the disease. We generalized the IPGS adding a statistical weight for each organ, through the transformation of Boolean features into "Boolean quantum features", inspired by the Quantum Mechanics. The organs' coefficients were set via the application of the genetic algorithm Pygad and, after that, we defined two new Integrated PolyGenic Score (IPGS1 and IPGS2). By applying a logistic regression with both IPGS2 (or indifferently IPGS1) and age as input, we reach an accuracy of 84-86%, thus improving the results previously shown in [16] by a factor of 10%.

Exome-wide association study to identify rare variants influencing COVID-19 outcomes: Results from the Host Genetics Initiative (preprint)

Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,048 severe disease cases and 571,009 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75-10.05, p=5.41x10-7). These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity. (preprint)

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 4,000 SARS-CoV-2-positive individuals were used to define an interpretable machine learning model for predicting COVID-19 severity. Firstly, 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. Noteworthly, 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.

Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis (preprint)

Host-expressed proteins on both host-cell and pathogen surfaces are widely exploited by pathogens, mediating cell entry (and exit) and influencing disease progression and transmission. This is highlighted by the diverse modes of coronavirus entry into cells and their consequent differing pathogenicity that is of direct relevance to the current SARS-CoV-2 pandemic. Host-expressed viral surface proteins bear post-translational modifications such as glycosylation that are essential for function but can confound or limit certain current biophysical methods used for dissecting key interactions. Several human coronaviruses attach to host cell-surface N-linked glycans that include forms of sialic acid. There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we show that novel protein NMR methods allow a complete and comprehensive analysis of the magnetization transfer caused by interactions between even heavily modified proteins and relevant ligands to generate quantitative binding data in a general manner. Our method couples direct, objective resonance-identification via a deconvolution algorithm with quantitative analysis using Bloch-McConnell equations to obtain interaction parameters (e.g. KD, kEx), which together enable structural modelling. By using an automated and openly available workflow, this method can be readily applied in a range of systems. This complete treatment of so-called 'saturation transfer' between protein and ligand now enables a general analysis of solution-phase ligand-protein binding beyond previously perceived limits of exchange rates, concentration or system - this allows 'universal' saturation transfer analysis (uSTA). uSTA proves critical in mapping direct interaction between natural sialoside sugar ligands and SARS-CoV-2-spike glycoprotein by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile spike-protein glycans (that also include sialosides). Using uSTA, 'end on'-binding by SARS-CoV-2-spike protein to sialoside glycan is revealed, which contrasts with an observed 'extended surface'-binding for previously validated heparin sugar ligands. Quantitative use of uSTA-derived restraints pinpoints likely binding modes to an intrinsically disordered region of the N-terminal domain of SARS-CoV-2-spike trimer. Consistent with this, glycan binding is minimally perturbed by antibodies that neutralize via binding the ACE2-binding domain (RBD) but strongly disrupted in the B1.1.7 and B1.351 variants-of-concern that possess hotspot mutations around the identified site. An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a possible model in which A-lineage-SARS-CoV-2 may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide a ready, quantitative method for widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males. (preprint)

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 autophagy and reduced TNF production was demonstrated in HEK293 cells transfected with TLR3-L412F 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 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.

Post-Mendelian genetic model in COVID-19 (preprint)

Host genetics is an emerging theme in COVID-19 and few common polymorphisms and some rare variants have been identified, either by GWAS or candidate gene approach, respectively. However, an organic model is still missing. Here, we propose a new model that takes into account common and rare germline variants applied in a cohort of 1,300 Italian SARS-CoV-2 positive individuals. Ordered logistic regression of clinical WHO grading on sex and age was used to obtain a binary phenotypic classification. Genetic variability from WES was synthesized in several boolean representations differentiated according to allele frequencies and genotype effect. LASSO logistic regression was used for extracting relevant genes. We defined about 100 common driver polymorphisms corresponding to classical "threshold model". Extracted genes were demonstrated to be gender specific. Stochastic rare more penetrant events on about additional 100 extracted genes, when occurred in a medium or severe background (common within the family), simulate Mendelian inheritance in 14% of subjects (having only 1 mutation) or oligogenic inheritance (in 10% having 2 mutations, in 11% having 3 mutations, etc). The combined effect of common and rare results can be described as an integrated polygenic score computed as: (nseverity - nmildness) + F (mseverity - mmildness) where n is the number of common driver genes, m is the number of driver rare variants and F is a factor for appropriately weighing the more powerful rare variants. We called the model "post-Mendelian". The model well describes the cohort, and patients are clustered in severe or mild by the integrated polygenic scores, the F factor being calibrated around 2, with a prediction capacity of 65% in males and 70% in females. In conclusion, this is the first comprehensive model interpreting host genetics in a holistic post-Mendelian manner. Further validations are needed in order to consolidate and refine the model which however holds true in thousands of SARS-CoV-2 Italian subjects.

Association of Toll-like receptor 7 variants with life-threatening COVID-19 disease in males (preprint)

Background: COVID-19 clinical presentation ranges from asymptomatic to fatal outcome. This variability is due in part to host genome specific mutations. Recently, two families in which COVID-19 segregates like an X-linked recessive monogenic disorder environmentally conditioned by SARS-CoV-2 have been reported leading to identification of loss-of-function variants in TLR7. Objective: We sought to determine whether the two families represent the tip of the iceberg of a subset of COVID-19 male patients. Methods: We compared male subjects with extreme phenotype selected from the Italian GEN-COVID cohort of 1178 SARS-CoV-2-infected subjects (<60y, 79 severe cases versus 77 control cases). We applied the LASSO Logistic Regression analysis, considering only rare variants on the young male subset, picking up TLR7 as the most important susceptibility gene. Results: Rare TLR7 missense variants were predicted to impact on protein function in severely affected males and in none of the asymptomatic subjects. We then investigated a similar white European cohort in Spain, confirming the impact of TRL7 variants. A gene expression profile analysis in peripheral blood mononuclear cells after stimulation with TLR7 agonist demonstrated a reduction of mRNA level of TLR7, IRF7, ISG15, IFN-[a] and IFN-{gamma} in COVID-19 patients compared with unaffected controls demonstrating an impairment in type I and II INF responses. Conclusion: Young males with TLR7 loss-of-function mutations and severe COVID-19 in the two reported families represent only a fraction of a broader and complex host genome situation. Specifically, missense mutations in the X-linked recessive TLR7 disorder may significantly contribute to disease susceptibility in up to 4% of severe COVID-19.

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in males. (preprint)

BackgroundCOVID-19 presentation ranges from asymptomatic to fatal. The variability in severity may be due in part to impaired Interferon type I response due to specific mutations in the host genome or to autoantibodies, explaining about 15% of the cases when combined. Exploring the host genome is thus warranted to further elucidate disease variability. MethodsWe developed a synthetic approach to genetic data representation using machine learning methods to investigate complementary genetic variability in COVID-19 infected patients that may explain disease severity, due to poly-amino acids repeat polymorphisms. Using host whole-exome sequencing data, we compared extreme phenotypic presentations (338 severe versus 300 asymptomatic cases) of the entire (men and women) Italian GEN-COVID cohort of 1178 subjects infected with SARS-CoV-2. We then applied the LASSO Logistic Regression model on Boolean gene-based representation of the poly-amino acids variability. FindingsShorter polyQ alleles ([≤]22) in the androgen receptor (AR) conferred protection against a more severe outcome in COVID-19 infection. In the subgroup of males with age <60 years, testosterone was higher in subjects with AR long-polyQ ([≥]23), possibly indicating receptor resistance (p=0.004 Mann-Whitney U test). Inappropriately low testosterone levels for the long-polyQ alleles 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 ([≥]23) and age>60 years had increased levels of C Reactive Protein (p=0.018). InterpretationOur results may contribute to design reliable clinical and public health measures and provide a rationale to test testosterone treatment as adjuvant therapy in symptomatic COVID-19 men expressing AR polyQ longer than 23 repeats. FundingMIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020). Private donors for COVID research and charity funds from Intesa San Paolo. BoxesO_ST_ABSEvidence before this studyC_ST_ABSWe searched on Medline, EMBASE, and Pubmed for articles published from January 2020 to August 2020 using various combinations of the search terms "sex-difference", "gender" AND SARS-Cov-2, or COVID. Epidemiological studies indicate that men and women are similarly infected by COVID-19, but the outcome is less favorable in men, independently of age. Several studies also showed that patients with hypogonadism tend to be more severely affected. A prompt intervention directed toward the most fragile subjects with SARS-Cov2 infection is currently the only strategy to reduce mortality. glucocorticoid treatment has been found cost-effective in improving the outcome of severe cases. Clinical algorithms have been proposed, but little is known on the ability of genetic profiling to predict outcome and disclose novel therapeutic strategies. Added-value of this studyIn a cohort of 1178 men and women with COVID-19, we used a supervised machine learning approach on a synthetic representation of the uncovered variability of the human genome due to poly-amino acid repeats. Comparing the genotype of patients with extreme manifestations (severe vs. asymptomatic), we found that the poly-glutamine repeat of the androgen receptor (AR) gene is relevant for COVID-19 disease and defective AR signaling identifies an association between male sex, testosterone exposure, and COVID-19 outcome. Failure of the endocrine feedback to overcome AR signaling defect by increasing testosterone levels during the infection leads to the fact that polyQ becomes dominant to T levels for the clinical outcome. Implications of all the available evidenceWe identify the first genetic polymorphism predisposing some men to develop a more severe disease irrespectively of age. Based on this, we suggest that sizing the AR poly-glutamine repeat has important implications in the diagnostic pipeline of patients affected by life-threatening COVID-19 infection. Most importantly, our studies open to the potential of using testosterone as adjuvant therapy for severe COVID-19 patients having defective androgen signaling, defined by this study as [≥]23 PolyQ repeats and inappropriate levels of circulating androgens.

Employing a Systematic Approach to Biobanking and Analyzing Genetic and Clinical Data for Advancing COVID-19 Research (preprint)

Within the GEN-COVID Multicenter Study, biospecimens from more than 1,000 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: i) severe multisystemic failure with either thromboembolic or pancreatic variant; ii) cytokine storm type, either severe with liver involvement or moderate; iii) moderate heart type, either with or without liver damage; iv) moderate multisystemic involvement, either with or without liver damage; v) mild, either with or without hyposmia. GCB and GCPR are further linked to the GEN-COVID Genetic Data Repository (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.