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.

Sustained IFN signaling is associated with delayed development of SARS-CoV-2-specific immunity (preprint)

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized COVID-19 patients. Integrated analysis using k-means reveal four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors are delineated by high and low antibody responses. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the Interferon paradox previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity.

Colocalization of expression transcripts with COVID-19 outcomes is rare across cell states, cell types and organs. (preprint)

Identifying causal genes at GWAS loci can help pinpoint targets for therapeutic interventions. Expression studies can disentangle such loci but signals from expression quantitative trait loci (eQTLs) often fail to colocalize-which means that the genetic control of measured expression is not shared with the genetic control of disease risk. This may be because gene expression is measured in the wrong cell type, physiological state, or organ. We tested whether Mendelian randomization (MR) could identify genes at loci influencing COVID-19 outcomes and whether the colocalization of genetic control of expression and COVID-19 outcomes was influenced by cell type, cell stimulation, and organ. We conducted MR of cis-eQTLs from single cell (scRNA-seq) and bulk RNA sequencing. We then tested variables that could influence colocalization, including cell type, cell stimulation, RNA sequencing modality, organ, symptoms of COVID-19, and SARS-CoV-2 status among individuals with symptoms of COVID-19. The outcomes used to test colocalization were COVID-19 severity and susceptibility as assessed in the Host Genetics Initiative release 7. Most transcripts identified using MR did not colocalize when tested across cell types, cell state and in different organs. Most that did colocalize likely represented false positives due to linkage disequilibrium. In general, colocalization was highly variable and at times inconsistent for the same transcript across cell type, cell stimulation and organ. While we identified factors that influenced colocalization for select transcripts, identifying 33 that mediate COVID-19 outcomes, our study suggests that colocalization of expression with COVID-19 outcomes is partially due to noisy signals even after following quality control and sensitivity testing. These findings illustrate the present difficulty of linking expression transcripts to disease outcomes and the need for skepticism when observing eQTL MR results, even accounting for cell types, stimulation state and different organs.

Aquaporin 3 modulates the risk of death conferred by dehydration in COVID-19 (preprint)

Severe COVID-19 has been associated with dehydration. Recently, a genetic variant near the aquaporin 3 (AQP3) water channel was associated with severe COVID-19 (rs60840586:G, Odds Ratio: 1.07, P=2.5*10-9). We show that dehydration is associated COVID-19 mortality (OR = 2.06 [95% CI = 1.62-2.65], P = 9.13*10-9), and is modulated by interaction with rs60840586:G genotype (OR = 1.95 [95% CI = 1.22-3.28], P = 0.0075).

Proteomic Characterization of Acute Kidney Injury in Patients Hospitalized with SARS-CoV2 Infection (preprint)

Background Acute kidney injury (AKI) is a known complication of COVID-19 and is associated with an increased risk of in-hospital mortality. Unbiased proteomics using biological specimens can lead to improved risk stratification and discover pathophysiological mechanisms. Methods Using measurements of ~4000 plasma proteins in two cohorts of patients hospitalized with COVID-19, we discovered and validated markers of COVID-associated AKI (stage 2 or 3) and long-term kidney dysfunction. In the discovery cohort (N= 437), we identified 413 higher plasma abundances of protein targets and 40 lower plasma abundances of protein targets associated with COVID-AKI (adjusted p <0.05). Of these, 62 proteins were validated in an external cohort (p <0.05, N =261). Results We demonstrate that COVID-AKI is associated with increased markers of tubular injury (NGAL) and myocardial injury. Using estimated glomerular filtration (eGFR) measurements taken after discharge, we also find that 25 of the 62 AKI-associated proteins are significantly associated with decreased post-discharge eGFR (adjusted p <0.05). Proteins most strongly associated with decreased post-discharge eGFR included desmocollin-2, trefoil factor 3, transmembrane emp24 domain-containing protein 10, and cystatin-Cindicating tubular dysfunction and injury. Conclusions Using clinical and proteomic data, our results suggest that while both acute and long-term COVID-associated kidney dysfunction are associated with markers of tubular dysfunction, AKI is driven by a largely multifactorial process involving hemodynamic instability and myocardial damage.

Alternative splicing in the lung influences COVID-19 severity and respiratory diseases. (preprint)

Hospital admission for COVID-19 remains common despite the successful development of vaccines and treatments. Thus, there is an ongoing need to identify targets for new COVID-19 therapies. Alternative splicing is an essential mechanism for generating functional diversity in protein isoforms and influences immune response to infection. However, the causal role of alternative splicing in COVID-19 severity and its potential therapeutic relevance is not fully understood. In this study, we evaluated the causal role of alternative splicing in COVID-19 severity and susceptibility using Mendelian randomization (MR). To do so, we performed two-sample MR to assess whether cis-sQTLs spanning 8,172 gene splicing in 5,295 genes were associated with COVID-19 outcomes in the COVID-19 Host Genetics Initiative, including up to 158,840 COVID-19 cases and 2,782,977 population controls. We identified that alternative splicing in lungs, rather than total RNA expression of OAS1, ATP11A, DPP9 and NPNT, was associated with COVID-19 severity. MUC1 splicing was associated with COVID-19 susceptibility. Further colocalization analyses supported a shared genetic mechanism between COVID-19 severity with idiopathic pulmonary fibrosis at ATP11A and DPP9 loci, and with chronic obstructive lung diseases at NPNT. We lastly showed that ATP11A, DPP9, NPNT, and MUC1 were highly expressed in lung alveolar epithelial cells, both in COVID-19 uninfected and infected samples. Taken together, these findings clarify the importance of alternative splicing of proteins in the lung for COVID-19 and other respiratory diseases, providing isoform-based targets for drug discovery.

Proteome-wide Mendelian randomization implicates nephronectin as an actionable mediator of the effect of obesity on COVID-19 severity (preprint)

Obesity is a major risk factor for COVID-19 severity; however, the underlying mechanism is not fully understood. Considering that obesity influences the human plasma proteome, we sought to identify circulating proteins mediating the effects of obesity on COVID-19 severity. We first screened 4,907 plasma proteins to identify proteins influenced by body mass index (BMI) using Mendelian randomization (MR). This yielded 1,216 proteins, whose effects on COVID-19 severity were assessed, again using MR. This two-step approach identified nephronectin (NPNT), for which a one standard deviation increase was associated with severe COVID-19 (odds ratio = 1.71, 95% CI: 1.45-2.02, P = 1.63 x 10-10). Colocalization analyses indicated that an NPNT splice isoform drove this effect. Overall, NPNT mediates 3.7% of the total effect of BMI on severe COVID-19. Finally, we found that decreasing body fat mass and increasing fat-free mass can lower NPNT levels and thus may improve COVID-19 outcomes. These findings provide actionable insights into how obesity influences COVID-19 severity.

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.

Circulating proteins to predict adverse COVID-19 outcomes (preprint)

Predicting COVID-19 severity is difficult, and the biological pathways involved are not fully understood. To approach this problem, we measured 4,701 circulating human protein abundances in two independent cohorts totaling 986 individuals. We then trained prediction models including protein abundances and clinical risk factors to predict adverse COVID-19 outcomes in 417 subjects and tested these models in a separate cohort of 569 individuals. For severe COVID-19, a baseline model including age and sex provided an area under the receiver operator curve (AUC) of 65% in the test cohort. Selecting 92 proteins from the 4,701 unique protein abundances improved the AUC to 88% in the training cohort, which remained relatively stable in the testing cohort at 86%, suggesting good generalizability. Proteins selected from different adverse COVID-19 outcomes were enriched for cytokine and cytokine receptors, but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of adverse COVID-19 outcomes. Further research is needed to understand how to incorporate protein measurement into clinical care.

Age-dependent impact of the major common genetic risk factor for COVID-19 on severity and mortality (preprint)

Background: There is considerable variability in COVID-19 outcomes amongst younger adults and some of this variation may be due to genetic predisposition. We characterized the clinical implications of the major genetic risk factor for COVID-19 severity, and its age-dependent effect, using individual-level data in a large international multi-centre consortium. Method: The major common COVID-19 genetic risk factor is a chromosome 3 locus, tagged by the marker rs10490770. We combined individual level data for 13,424 COVID-19 positive patients (N=6,689 hospitalized) from 17 cohorts in nine countries to assess the association of this genetic marker with mortality, COVID-19-related complications and laboratory values. We next examined if the magnitude of these associations varied by age and were independent from known clinical COVID-19 risk factors. Findings: We found that rs10490770 risk allele carriers experienced an increased risk of all-cause mortality (hazard ratio [HR] 1.4, 95% confidence interval [CI] 1.2-1.6) and COVID-19 related mortality (HR 1.5, 95%CI 1.3-1.8). Risk allele carriers had increased odds of several COVID-19 complications: severe respiratory failure (odds ratio [OR] 2.0, 95%CI 1.6-2.6), venous thromboembolism (OR 1.7, 95%CI 1.2-2.4), and hepatic injury (OR 1.6, 95%CI 1.2-2.0). Risk allele carriers [≤] 60 years had higher odds of death or severe respiratory failure (OR 2.6, 95%CI 1.8-3.9) compared to those > 60 years OR 1.5 (95%CI 1.3-1.9, interaction p-value=0.04). Amongst individuals [≤] 60 years who died or experienced severe respiratory COVID-19 outcome, we found that 31.8% (95%CI 27.6-36.2) were risk variant carriers, compared to 13.9% (95%CI 12.6-15.2%) of those not experiencing these outcomes. Prediction of death or severe respiratory failure among those [≤] 60 years improved when including the risk allele (AUC 0.82 vs 0.84, p=0.016) and the prediction ability of rs10490770 risk allele was similar to, or better than, most established clinical risk factors. Interpretation: The major common COVID-19 risk locus on chromosome 3 is associated with increased risks of morbidity and mortality and these are more pronounced amongst individuals [≤] 60 years. The effect on COVID-19 severity was similar to, or larger than most established risk factors, suggesting potential implications for clinical risk management. Funding: Funding was obtained by each of the participating cohorts individually.

Failure to replicate the association of rare loss-of-function variants in type I IFN immunity genes with severe COVID-19 (preprint)

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2 (preprint)

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic 'priming' (S1/S2) and 'fusion-activation' (S2') for viral entry. The S-protein furin-like motifs PRRAR685{downarrow} and KPSKR815{downarrow} indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2' processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (S1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2', but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1', activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and S1/S2 severely curtail 'pH-independent' viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2

SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome (preprint)

The Coronavirus Disease 2019 (COVID-19) pandemic has caused millions of deaths and will continue to exact incalculable tolls worldwide. While great strides have been made toward understanding and combating the mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, relatively little is known about the individual SARS-CoV-2 proteins that contribute to pathogenicity during infection and that cause neurological sequela after viral clearance. We used Drosophila to develop an in vivo model that characterizes mechanisms of SARS-CoV-2 pathogenicity, and found ORF3a adversely affects longevity and motor function by inducing apoptosis and inflammation in the nervous system. Chloroquine alleviated ORF3a induced phenotypes in the CNS, arguing our Drosophila model is amenable to high throughput drug screening. Our work provides novel insights into the pathogenic nature of SARS-CoV-2 in the nervous system that can be used to develop new treatment strategies for post-viral syndrome.

Vitamin D and Covid-19 Susceptibility and Severity: a Mendelian Randomization Study (preprint)

Introduction Increased vitamin D levels, as reflected by 25OHD measurements, has been proposed to protect against Covid-19 disease based on in-vitro, observational, and ecological studies. However, vitamin D levels are associated with many confounding variables and thus associations described to date may not be causal. Vitamin D MR studies have provided results that are concordant with large-scale vitamin D randomized trials. Here, we used two-sample MR to assess the effect of circulating 25OHD levels on Covid-19 susceptibility. Methods Genetic variants strongly associated with 25OHD levels in a 443,734-participant genome-wide association study (GWAS) were used as instrumental variables. GWASs of Covid-19 susceptibility and severity from the Covid-19 Host Genetics Initiative were used to test the effect of 25OHD levels on these outcomes. Cohorts from the Covid-19 Host Genetics Initiative GWAS included up to 966,395 individuals of European ancestry. Results Genetically increased 25OHD levels by one standard deviation on the logarithmic scale had no clear effect on susceptibility but tended to increase the odds ratio of hospitalization (OR = 2.34; 95% CI: 1.33, 4.11) and severe disease (OR = 2.21; 95% CI: 0.87, 5.55). Extensive sensitivity analyses probing the assumptions of MR provided consistent estimates. Conclusion These findings do not support a protective role of increased 25OHD levels on Covid-19 outcomes and may suggest harm. At present, individuals should not use vitamin D supplements to protect against Covid-19 outcomes, and on-going supplementation trials should closely monitor for signals of harm.