An explainable model of host genetic interactions linked to COVID-19 severity

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.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

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

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

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

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Expression of recombinant olive lyase hydroperoxide in Escherichia coli and Pichia pastoris yeast Study of structure-function relationships of the enzyme

L’hydroperoxyde lyase (HPL) est une enzyme présente chez les végétaux supérieurs et impliquée dans la voie de la lipoxygénase. Elle agit sur les 9- et/ou les 13-hydroperoxydes d’acide gras pour produire des aldéhydes à 6 ou 9 carbones, responsables de l’odeur fraîche de l’herbe coupée dite « note verte », et très utilisés par les industries cosmétiques et agroalimentaires. Afin de répondre à la demande croissante en molécules à note verte naturelles, des systèmes de production utilisant des enzymes recombinantes sont développés. Ainsi, l’HPL d’olive (HPLwt) et l’enzyme dépourvue de son peptide de transit chloroplastique (HPLdel), ont été clonées au laboratoire et exprimées chez Escherichia coli. Les deux enzymes recombinantes ont été purifiées puis caractérisées biochimiquement. Elles sont spécifiques des 13-hydroperoxydes, présentent une activité maximale à pH 7,5 et à 25°C, et agissent plus efficacement sur le 13-hydroperoxyde d’acide linolénique. L’étude de la stabilité de l’HPLwt et de l’HPLdel au cours du temps a montré que la totalité de l’activité enzymatique est conservée durant cinq semaines de stockage à -80°C en présence de glycérol 10%. De plus, l’ajout de composés chimiques tels que le NaCl, le Na2SO4 et la glycine ont permis d’augmenter l’activité des deux enzymes. Parallèlement, les HPL recombinantes ont été exprimées dans un système eucaryote, la levure Pichia pastoris. Les enzymes ne sont pas sécrétées dans le milieu de culture, mais exprimées au niveau intracellulaire, avec une activité maximale obtenue après 24h d’induction. Par ailleurs, les relations structure-fonction de l’HPL ont été étudiées. La structure tridimensionnelle de la 13-HPL d’olive a été modélisée grâce à des méthodes computationnelles. Des acides aminés potentiellement impliqués dans la régiospécificité de l’enzyme ont été ciblés, puis modifiés par mutagénèse dirigée. Ainsi, le mutant T302K présente une efficacité catalytique sur les 13-hydroperoxydes similaire à celle de l’HPLwt, mais il est également actif sur le 9-hydroperoxyde d’acide linolénique.Hydroperoxide lyase (HPL) is an enzyme found in higher plants and involved in the lipoxygenase pathway. HPL acts on the 9- and/or 13-hydroperoxy fatty acids to produce C6 or C9 aldehydes, responsible for the fresh odor of cut grass known as "green note" and widely used by cosmetic and food industries. Production systems using recombinant enzymes are developed to meet the growing demand for natural green leaf volatiles. Thus, HPL from olive fruit (HPLwt) and the enzyme with its chloroplast transit peptide deleted (HPLdel), were cloned in the laboratory and expressed in Escherichia coli. Both recombinant enzymes were purified and biochemically characterized. They act only on 13-hydroperoxides, exhibit maximum activity at pH 7.5 and 25°C, and are more effective on the 13-hydroperoxy linoleic acid. The study of HPLwt and HPLdel stability over time has shown that all of the enzymatic activity is retained during a five weeks storage at -80°C in the presence of 10% glycerol. Furthermore, the addition of chemical compounds such as NaCl, Na2SO4 and glycine have increased the activity of both enzymes. Meanwhile, recombinant HPLs have been expressed in the eukaryotic system Pichia pastoris yeast. The HPLs are not secreted into the culture medium, but are expressed intracellularly, with a maximum after 24h of induction. Moreover, HPL structure-function relations were studied. The olive 13-HPL three dimensional structure has been built by computational modelling. Amino acids potentially involved in regiospecificity of the enzyme have been identified and modified by site-directed mutagenesis. Thus, T302K mutant has a catalytic efficiency on 13-hydroperoxides similar to the HPLwt, but it is also active on the 9-hydroperoxide of linoleic acid

Biocatalytic Synthesis of Natural Green Leaf Volatiles Using the Lipoxygenase Metabolic Pathway

In higher plants, the lipoxygenase enzymatic pathway combined actions of several enzymes to convert lipid substrates into signaling and defense molecules called phytooxylipins including short chain volatile aldehydes, alcohols, and esters, known as green leaf volatiles (GLVs). GLVs are synthesized from C18:2 and C18:3 fatty acids that are oxygenated by lipoxygenase (LOX) to form corresponding hydroperoxides, then the action of hydroperoxide lyase (HPL) produces C6 or C9 aldehydes that can undergo isomerization, dehydrogenation, and esterification. GLVs are commonly used as flavors to confer a fresh green odor of vegetable to perfumes, cosmetics, and food products. Given the increasing demand in these natural flavors, biocatalytic processes using the LOX pathway reactions constitute an interesting application. Vegetable oils, chosen for their lipid profile are converted in natural GLVs with high added value. This review describes the enzymatic reactions of GLVs biosynthesis in the plant, as well as the structural and functional properties of the enzymes involved. The various stages of the biocatalytic production processes are approached from the lipid substrate to the corresponding aldehyde or alcoholic aromas, as well as the biotechnological improvements to enhance the production potential of the enzymatic catalysts

Expression de l’hydroperoxyde lyase recombinante d’olive chez la bactérie Escherichia coli et chez la levure Pichia pastoris Etude des relations structure-fonction de l’enzyme

Hydroperoxide lyase (HPL) is an enzyme found in higher plants and involved in the lipoxygenase pathway. HPL acts on the 9- and/or 13-hydroperoxy fatty acids to produce C6 or C9 aldehydes, responsible for the fresh odor of cut grass known as "green note" and widely used by cosmetic and food industries. Production systems using recombinant enzymes are developed to meet the growing demand for natural green leaf volatiles. Thus, HPL from olive fruit (HPLwt) and the enzyme with its chloroplast transit peptide deleted (HPLdel), were cloned in the laboratory and expressed in Escherichia coli. Both recombinant enzymes were purified and biochemically characterized. They act only on 13-hydroperoxides, exhibit maximum activity at pH 7.5 and 25°C, and are more effective on the 13-hydroperoxy linoleic acid. The study of HPLwt and HPLdel stability over time has shown that all of the enzymatic activity is retained during a five weeks storage at -80°C in the presence of 10% glycerol. Furthermore, the addition of chemical compounds such as NaCl, Na2SO4 and glycine have increased the activity of both enzymes. Meanwhile, recombinant HPLs have been expressed in the eukaryotic system Pichia pastoris yeast. The HPLs are not secreted into the culture medium, but are expressed intracellularly, with a maximum after 24h of induction. Moreover, HPL structure-function relations were studied. The olive 13-HPL three dimensional structure has been built by computational modelling. Amino acids potentially involved in regiospecificity of the enzyme have been identified and modified by site-directed mutagenesis. Thus, T302K mutant has a catalytic efficiency on 13-hydroperoxides similar to the HPLwt, but it is also active on the 9-hydroperoxide of linoleic acid.L’hydroperoxyde lyase (HPL) est une enzyme présente chez les végétaux supérieurs et impliquée dans la voie de la lipoxygénase. Elle agit sur les 9- et/ou les 13-hydroperoxydes d’acide gras pour produire des aldéhydes à 6 ou 9 carbones, responsables de l’odeur fraîche de l’herbe coupée dite « note verte », et très utilisés par les industries cosmétiques et agroalimentaires. Afin de répondre à la demande croissante en molécules à note verte naturelles, des systèmes de production utilisant des enzymes recombinantes sont développés. Ainsi, l’HPL d’olive (HPLwt) et l’enzyme dépourvue de son peptide de transit chloroplastique (HPLdel), ont été clonées au laboratoire et exprimées chez Escherichia coli. Les deux enzymes recombinantes ont été purifiées puis caractérisées biochimiquement. Elles sont spécifiques des 13-hydroperoxydes, présentent une activité maximale à pH 7,5 et à 25°C, et agissent plus efficacement sur le 13-hydroperoxyde d’acide linolénique. L’étude de la stabilité de l’HPLwt et de l’HPLdel au cours du temps a montré que la totalité de l’activité enzymatique est conservée durant cinq semaines de stockage à -80°C en présence de glycérol 10%. De plus, l’ajout de composés chimiques tels que le NaCl, le Na2SO4 et la glycine ont permis d’augmenter l’activité des deux enzymes. Parallèlement, les HPL recombinantes ont été exprimées dans un système eucaryote, la levure Pichia pastoris. Les enzymes ne sont pas sécrétées dans le milieu de culture, mais exprimées au niveau intracellulaire, avec une activité maximale obtenue après 24h d’induction. Par ailleurs, les relations structure-fonction de l’HPL ont été étudiées. La structure tridimensionnelle de la 13-HPL d’olive a été modélisée grâce à des méthodes computationnelles. Des acides aminés potentiellement impliqués dans la régiospécificité de l’enzyme ont été ciblés, puis modifiés par mutagénèse dirigée. Ainsi, le mutant T302K présente une efficacité catalytique sur les 13-hydroperoxydes similaire à celle de l’HPLwt, mais il est également actif sur le 9-hydroperoxyde d’acide linolénique

Expression de l’hydroperoxyde lyase recombinante d’olive chez la bactérie Escherichia coli et chez la levure Pichia pastoris Etude des relations structure-fonction de l’enzyme

Hydroperoxide lyase (HPL) is an enzyme found in higher plants and involved in the lipoxygenase pathway. HPL acts on the 9- and/or 13-hydroperoxy fatty acids to produce C6 or C9 aldehydes, responsible for the fresh odor of cut grass known as "green note" and widely used by cosmetic and food industries. Production systems using recombinant enzymes are developed to meet the growing demand for natural green leaf volatiles. Thus, HPL from olive fruit (HPLwt) and the enzyme with its chloroplast transit peptide deleted (HPLdel), were cloned in the laboratory and expressed in Escherichia coli. Both recombinant enzymes were purified and biochemically characterized. They act only on 13-hydroperoxides, exhibit maximum activity at pH 7.5 and 25°C, and are more effective on the 13-hydroperoxy linoleic acid. The study of HPLwt and HPLdel stability over time has shown that all of the enzymatic activity is retained during a five weeks storage at -80°C in the presence of 10% glycerol. Furthermore, the addition of chemical compounds such as NaCl, Na2SO4 and glycine have increased the activity of both enzymes. Meanwhile, recombinant HPLs have been expressed in the eukaryotic system Pichia pastoris yeast. The HPLs are not secreted into the culture medium, but are expressed intracellularly, with a maximum after 24h of induction. Moreover, HPL structure-function relations were studied. The olive 13-HPL three dimensional structure has been built by computational modelling. Amino acids potentially involved in regiospecificity of the enzyme have been identified and modified by site-directed mutagenesis. Thus, T302K mutant has a catalytic efficiency on 13-hydroperoxides similar to the HPLwt, but it is also active on the 9-hydroperoxide of linoleic acid.L’hydroperoxyde lyase (HPL) est une enzyme présente chez les végétaux supérieurs et impliquée dans la voie de la lipoxygénase. Elle agit sur les 9- et/ou les 13-hydroperoxydes d’acide gras pour produire des aldéhydes à 6 ou 9 carbones, responsables de l’odeur fraîche de l’herbe coupée dite « note verte », et très utilisés par les industries cosmétiques et agroalimentaires. Afin de répondre à la demande croissante en molécules à note verte naturelles, des systèmes de production utilisant des enzymes recombinantes sont développés. Ainsi, l’HPL d’olive (HPLwt) et l’enzyme dépourvue de son peptide de transit chloroplastique (HPLdel), ont été clonées au laboratoire et exprimées chez Escherichia coli. Les deux enzymes recombinantes ont été purifiées puis caractérisées biochimiquement. Elles sont spécifiques des 13-hydroperoxydes, présentent une activité maximale à pH 7,5 et à 25°C, et agissent plus efficacement sur le 13-hydroperoxyde d’acide linolénique. L’étude de la stabilité de l’HPLwt et de l’HPLdel au cours du temps a montré que la totalité de l’activité enzymatique est conservée durant cinq semaines de stockage à -80°C en présence de glycérol 10%. De plus, l’ajout de composés chimiques tels que le NaCl, le Na2SO4 et la glycine ont permis d’augmenter l’activité des deux enzymes. Parallèlement, les HPL recombinantes ont été exprimées dans un système eucaryote, la levure Pichia pastoris. Les enzymes ne sont pas sécrétées dans le milieu de culture, mais exprimées au niveau intracellulaire, avec une activité maximale obtenue après 24h d’induction. Par ailleurs, les relations structure-fonction de l’HPL ont été étudiées. La structure tridimensionnelle de la 13-HPL d’olive a été modélisée grâce à des méthodes computationnelles. Des acides aminés potentiellement impliqués dans la régiospécificité de l’enzyme ont été ciblés, puis modifiés par mutagénèse dirigée. Ainsi, le mutant T302K présente une efficacité catalytique sur les 13-hydroperoxydes similaire à celle de l’HPLwt, mais il est également actif sur le 9-hydroperoxyde d’acide linolénique