Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended genome-wide association meta-analysis of a well-characterized cohort of 3255 COVID-19 patients with respiratory failure and 12 488 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a ~0.9-Mb inversion polymorphism that creates two highly differentiated haplotypes and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative including non-Caucasian individuals, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.S.E.H. and C.A.S. partially supported genotyping through a philanthropic donation. A.F. and D.E. were supported by a grant from the German Federal Ministry of Education and COVID-19 grant Research (BMBF; ID:01KI20197); A.F., D.E. and F.D. were supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). D.E. was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the Computational Life Sciences funding concept (CompLS grant 031L0165). D.E., K.B. and S.B. acknowledge the Novo Nordisk Foundation (NNF14CC0001 and NNF17OC0027594). T.L.L., A.T. and O.Ö. were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project numbers 279645989; 433116033; 437857095. M.W. and H.E. are supported by the German Research Foundation (DFG) through the Research Training Group 1743, ‘Genes, Environment and Inflammation’. L.V. received funding from: Ricerca Finalizzata Ministero della Salute (RF-2016-02364358), Italian Ministry of Health ‘CV PREVITAL’—strategie di prevenzione primaria cardiovascolare primaria nella popolazione italiana; The European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- and for the project ‘REVEAL’; Fondazione IRCCS Ca’ Granda ‘Ricerca corrente’, Fondazione Sviluppo Ca’ Granda ‘Liver-BIBLE’ (PR-0391), Fondazione IRCCS Ca’ Granda ‘5permille’ ‘COVID-19 Biobank’ (RC100017A). A.B. was supported by a grant from Fondazione Cariplo to Fondazione Tettamanti: ‘Bio-banking of Covid-19 patient samples to support national and international research (Covid-Bank). This research was partly funded by an MIUR grant to the Department of Medical Sciences, under the program ‘Dipartimenti di Eccellenza 2018–2022’. This study makes use of data generated by the GCAT-Genomes for Life. Cohort study of the Genomes of Catalonia, Fundació IGTP (The Institute for Health Science Research Germans Trias i Pujol) IGTP is part of the CERCA Program/Generalitat de Catalunya. GCAT is supported by Acción de Dinamización del ISCIII-MINECO and the Ministry of Health of the Generalitat of Catalunya (ADE 10/00026); the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR 529). M.M. received research funding from grant PI19/00335 Acción Estratégica en Salud, integrated in the Spanish National RDI Plan and financed by ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (European Regional Development Fund (FEDER)-Una manera de hacer Europa’). B.C. is supported by national grants PI18/01512. X.F. is supported by the VEIS project (001-P-001647) (co-funded by the European Regional Development Fund (ERDF), ‘A way to build Europe’). Additional data included in this study were obtained in part by the COVICAT Study Group (Cohort Covid de Catalunya) supported by IsGlobal and IGTP, European Institute of Innovation & Technology (EIT), a body of the European Union, COVID-19 Rapid Response activity 73A and SR20-01024 La Caixa Foundation. A.J. and S.M. were supported by the Spanish Ministry of Economy and Competitiveness (grant numbers: PSE-010000-2006-6 and IPT-010000-2010-36). A.J. was also supported by national grant PI17/00019 from the Acción Estratégica en Salud (ISCIII) and the European Regional Development Fund (FEDER). The Basque Biobank, a hospital-related platform that also involves all Osakidetza health centres, the Basque government’s Department of Health and Onkologikoa, is operated by the Basque Foundation for Health Innovation and Research-BIOEF. M.C. received Grants BFU2016-77244-R and PID2019-107836RB-I00 funded by the Agencia Estatal de Investigación (AEI, Spain) and the European Regional Development Fund (FEDER, EU). M.R.G., J.A.H., R.G.D. and D.M.M. are supported by the ‘Spanish Ministry of Economy, Innovation and Competition, the Instituto de Salud Carlos III’ (PI19/01404, PI16/01842, PI19/00589, PI17/00535 and GLD19/00100) and by the Andalussian government (Proyectos Estratégicos-Fondos Feder PE-0451-2018, COVID-Premed, COVID GWAs). The position held by Itziar de Rojas Salarich is funded by grant FI20/00215, PFIS Contratos Predoctorales de Formación en Investigación en Salud. Enrique Calderón’s team is supported by CIBER of Epidemiology and Public Health (CIBERESP), ‘Instituto de Salud Carlos III’. J.C.H. reports grants from Research Council of Norway grant no 312780 during the conduct of the study. E.S. reports grants from Research Council of Norway grant no. 312769. The BioMaterialBank Nord is supported by the German Center for Lung Research (DZL), Airway Research Center North (ARCN). The BioMaterialBank Nord is member of popgen 2.0 network (P2N). P.K. Bergisch Gladbach, Germany and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. He is supported by the German Federal Ministry of Education and Research (BMBF). O.A.C. is supported by the German Federal Ministry of Research and Education and is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—CECAD, EXC 2030–390661388. The COMRI cohort is funded by Technical University of Munich, Munich, Germany. This work was supported by grants of the Rolf M. Schwiete Stiftung, the Saarland University, BMBF and The States of Saarland and Lower Saxony. K.U.L. is supported by the German Research Foundation (DFG, LU-1944/3-1). Genotyping for the BoSCO study is funded by the Institute of Human Genetics, University Hospital Bonn. F.H. was supported by the Bavarian State Ministry for Science and Arts. Part of the genotyping was supported by a grant to A.R. from the German Federal Ministry of Education and Research (BMBF, grant: 01ED1619A, European Alzheimer DNA BioBank, EADB) within the context of the EU Joint Programme—Neurodegenerative Disease Research (JPND). Additional funding was derived from the German Research Foundation (DFG) grant: RA 1971/6-1 to A.R. P.R. is supported by the DFG (CCGA Sequencing Centre and DFG ExC2167 PMI and by SH state funds for COVID19 research). F.T. is supported by the Clinician Scientist Program of the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). C.L. and J.H. are supported by the German Center for Infection Research (DZIF). T.B., M.M.B., O.W. und A.H. are supported by the Stiftung Universitätsmedizin Essen. M.A.-H. was supported by Juan de la Cierva Incorporacion program, grant IJC2018-035131-I funded by MCIN/AEI/10.13039/501100011033. E.C.S. is supported by the Deutsche Forschungsgemeinschaft (DFG; SCHU 2419/2-1).Peer reviewe

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic ∼0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.Andre Franke and David Ellinghaus were supported by a grant from the German Federal Ministry of Education and Research (01KI20197), Andre Franke, David Ellinghaus and Frauke Degenhardt were supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence “Precision Medicine in Chronic Inflammation” (EXC2167). David Ellinghaus was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the Computational Life Sciences funding concept (CompLS grant 031L0165). David Ellinghaus, Karina Banasik and Søren Brunak acknowledge the Novo Nordisk Foundation (grant NNF14CC0001 and NNF17OC0027594). Tobias L. Lenz, Ana Teles and Onur Özer were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project numbers 279645989; 433116033; 437857095. Mareike Wendorff and Hesham ElAbd are supported by the German Research Foundation (DFG) through the Research Training Group 1743, "Genes, Environment and Inflammation". This project was supported by a Covid-19 grant from the German Federal Ministry of Education and Research (BMBF; ID: 01KI20197). Luca Valenti received funding from: Ricerca Finalizzata Ministero della Salute RF2016-02364358, Italian Ministry of Health ""CV PREVITAL – strategie di prevenzione primaria cardiovascolare primaria nella popolazione italiana; The European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- and for the project ""REVEAL""; Fondazione IRCCS Ca' Granda ""Ricerca corrente"", Fondazione Sviluppo Ca' Granda ""Liver-BIBLE"" (PR-0391), Fondazione IRCCS Ca' Granda ""5permille"" ""COVID-19 Biobank"" (RC100017A). Andrea Biondi was supported by the grant from Fondazione Cariplo to Fondazione Tettamanti: "Biobanking of Covid-19 patient samples to support national and international research (Covid-Bank). This research was partly funded by a MIUR grant to the Department of Medical Sciences, under the program "Dipartimenti di Eccellenza 2018–2022". This study makes use of data generated by the GCAT-Genomes for Life. Cohort study of the Genomes of Catalonia, Fundació IGTP. IGTP is part of the CERCA Program / Generalitat de Catalunya. GCAT is supported by Acción de Dinamización del ISCIIIMINECO and the Ministry of Health of the Generalitat of Catalunya (ADE 10/00026); the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR 529). Marta Marquié received research funding from ant PI19/00335 Acción Estratégica en Salud, integrated in the Spanish National RDI Plan and financed by ISCIIISubdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER-Una manera de hacer Europa").Beatriz Cortes is supported by national grants PI18/01512. Xavier Farre is supported by VEIS project (001-P-001647) (cofunded by European Regional Development Fund (ERDF), “A way to build Europe”). Additional data included in this study was obtained in part by the COVICAT Study Group (Cohort Covid de Catalunya) supported by IsGlobal and IGTP, EIT COVID-19 Rapid Response activity 73A and SR20-01024 La Caixa Foundation. Antonio Julià and Sara Marsal were supported by the Spanish Ministry of Economy and Competitiveness (grant numbers: PSE-010000-2006-6 and IPT-010000-2010-36). Antonio Julià was also supported the by national grant PI17/00019 from the Acción Estratégica en Salud (ISCIII) and the FEDER. The Basque Biobank is a hospitalrelated platform that also involves all Osakidetza health centres, the Basque government's Department of Health and Onkologikoa, is operated by the Basque Foundation for Health Innovation and Research-BIOEF. Mario Cáceres received Grants BFU2016-77244-R and PID2019-107836RB-I00 funded by the Agencia Estatal de Investigación (AEI, Spain) and the European Regional Development Fund (FEDER, EU). Manuel Romero Gómez, Javier Ampuero Herrojo, Rocío Gallego Durán and Douglas Maya Miles are supported by the “Spanish Ministry of Economy, Innovation and Competition, the Instituto de Salud Carlos III” (PI19/01404, PI16/01842, PI19/00589, PI17/00535 and GLD19/00100), and by the Andalussian government (Proyectos Estratégicos-Fondos Feder PE-0451-2018, COVID-Premed, COVID GWAs). The position held by Itziar de Rojas Salarich is funded by grant FI20/00215, PFIS Contratos Predoctorales de Formación en Investigación en Salud. Enrique Calderón's team is supported by CIBER of Epidemiology and Public Health (CIBERESP), "Instituto de Salud Carlos III". Jan Cato Holter reports grants from Research Council of Norway grant no 312780 during the conduct of the study. Dr. Solligård: reports grants from Research Council of Norway grant no 312769. The BioMaterialBank Nord is supported by the German Center for Lung Research (DZL), Airway Research Center North (ARCN). The BioMaterialBank Nord is member of popgen 2.0 network (P2N). Philipp Koehler has received non-financial scientific grants from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. He is supported by the German Federal Ministry of Education and Research (BMBF).Oliver A. Cornely is supported by the German Federal Ministry of Research and Education and is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388. The COMRI cohort is funded by Technical University of Munich, Munich, Germany. Genotyping was performed by the Genotyping laboratory of Institute for Molecular Medicine Finland FIMM Technology Centre, University of Helsinki. This work was supported by grants of the Rolf M. Schwiete Stiftung, the Saarland University, BMBF and The States of Saarland and Lower Saxony. Kerstin U. Ludwig is supported by the German Research Foundation (DFG, LU-1944/3-1). Genotyping for the BoSCO study is funded by the Institute of Human Genetics, University Hospital Bonn. Frank Hanses was supported by the Bavarian State Ministry for Science and Arts. Part of the genotyping was supported by a grant to Alfredo Ramirez from the German Federal Ministry of Education and Research (BMBF, grant: 01ED1619A, European Alzheimer DNA BioBank, EADB) within the context of the EU Joint Programme – Neurodegenerative Disease Research (JPND). Additional funding was derived from the German Research Foundation (DFG) grant: RA 1971/6-1 to Alfredo Ramirez. Philip Rosenstiel is supported by the DFG (CCGA Sequencing Centre and DFG ExC2167 PMI and by SH state funds for COVID19 research). Florian Tran is supported by the Clinician Scientist Program of the Deutsche Forschungsgemeinschaft Cluster of Excellence “Precision Medicine in Chronic Inflammation” (EXC2167). Christoph Lange and Jan Heyckendorf are supported by the German Center for Infection Research (DZIF). Thorsen Brenner, Marc M Berger, Oliver Witzke und Anke Hinney are supported by the Stiftung Universitätsmedizin Essen. Marialbert Acosta-Herrera was supported by Juan de la Cierva Incorporacion program, grant IJC2018-035131-I funded by MCIN/AEI/10.13039/501100011033. Eva C Schulte is supported by the Deutsche Forschungsgemeinschaft (DFG; SCHU 2419/2-1).N

Complejidad de la cuasiespecie del virus de la hepatitis B en la región X/preCore: asociación con la evolución de la infección con y sin tratamiento antiviral

El Virus de la hepatitis B (VHB) tiene una tasa de mutación de 3,2×10-5 – 7,9×10-5 nucleótidos sustituidos/ciclo replicativo, esto se debe a que este ciclo consta de un paso de retrotranscripción llevado a cabo por una transcriptasa reversa que carece de actividad correctora de errores exonucleasa 3’-5’. Este hecho resulta en una enorme diversidad genética en forma de genotipos, subgenotipos, cuasiespecies (QS) y variantes en diferentes regiones del genoma. Existen numerosas evidencias de la relación entre la diversidad genética del VHB, e incluso de la complejidad de la QS, y la tasa de seroconversión HBeAg, los niveles de ADN-VHB, el escape inmune, la patogénesis y la respuesta al tratamiento, lo que pone de manifiesto la relevancia de estudiar dicha variabilidad y complejidad. El objetivo de este proyecto de tesis fue evaluar la complejidad de la QS en la región preCore/Core y su relación con el estatus HBeAg, y analizar los cambios en la QS durante la dinámica natural y bajo el tratamiento con análogos de nucleós(t)idos (NUCs), a través de los parámetros de complejidad (entropía de Shannon normalizada, frecuencia de mutación y diversidad nucleotídica). El segundo objetivo fue estudiar la complejidad a nivel de genotipos mediante la evaluación de la presencia de mezclas y sus consecuencias, en dos regiones del genoma (polimerasa/superficie y X/preCore) en pacientes con infección crónica, también durante dos períodos (dinámica natural y tratamiento con NUCs). Para ello en el primer estudio se llevó a cabo la secuenciación masiva por UDPS (ultra-deep pyrosequencing) de la región preCore/Core en muestras secuenciales (en el momento del diagnóstico, tras un tiempo sin tratamiento y después de no responder al mismo) de 10 pacientes seleccionados y agrupados según su estatus HBeAg. En el segundo estudio se analizaron muestras de otros 10 pacientes en la región polimerasa/superficie y X/preCore en una muestra basal, tras un tiempo sin tratamiento y durante el tratamiento, el genotipo se determinó por filogénesis. Los resultados del primer estudio revelan mayor complejidad de la QS en pacientes HBeAg(-) que HBeAg(+), confirmando estudios previos. Los resultados también muestran que la alta complejidad en la región preCore está asociada con una baja replicación, en concordancia con el papel clave de esta región en la misma, y sugiere una respuesta inmune aumentada en los pacientes HBeAg(-), probablemente debido a la falta de actividad inmunomoduladora de HBeAg. La selección positiva de variantes core en pacientes HBeAg o fluctuante que se ha observado, puede ser entendida como un mecanismo potencial de escape del sistema inmune por cambios en la secuencia de la nucleocápside. Por último, se ha encontrado una correlación negativa de la evolución de la QS en el período sin tratamiento y en el período con tratamiento, lo que sugiere la importancia de estudiar la complejidad de la QS antes de tratar a los pacientes como un posible factor predictivo de la evolución del virus en el caso de no respondedores a NUCs. Los resultados del segundo estudio evidencian infecciones con mezclas de genotipos y su cambio a lo largo del tiempo y ponen de manifiesto la compleja dinámica de la QS del VHB como un mecanismo adicional para adaptarse a nuevas situaciones, como pueden ser la repuesta del sistema inmune o un determinado tratamiento antiviral. Se han encontrado discrepancias entre los genotipos obtenidos en la región polimerasa/superficie y X/preCore que sugieren un fenómeno de recombinación intergenotípica y que señalan la necesidad de consensuar una región y técnica para el genotipaje, que lleve a una mejor comprensión del significado clínico de la clasificación genotípica.Hepatitis B Virus (HBV) shows a high mutation rate (3.2 × 10-5 - 7.9 × 10-5 nucleotide substitutions/replicative cycle), due to its unique life cycle which requires an error-prone reverse transcriptase for replication. This fact results in a tremendous genetic variation in the form of genotypes, sub-genotypes, quasispecies (QA) and variants in different regions of the genome. There has been considerable evidence on the relationship between HBV genetic variation, or even QA complexity, and HBeAg seroconversion, HBV-DNA levels, immune scape, pathogenesis and treatment response, showing the relevance of the analysis of this variability and complexity. The aim of this thesis project was to evaluate HBV QA complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUCs) treatment, measuring complexity parameters (normalized Shannon entropy, mutation frequency and nucleotide diversity). The second aim was to study the complexity at genotype level by assessing the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions in patients with chronic hepatitis B virus, also during two periods (natural evaluation and NUCs treatment). For this purpose ultra-deep pyrosequencing (UDPS) of HBV preCore/Core regions in sequential samples (baseline at moment of diagnosis, after a period treatment-free, and after a period of treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status, were analyzed in the first study. In the second study, thirty samples from another ten chronic hepatitis B patients were analyzed in the polymerase/surface and X/preCore regions in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment and the HBV genotype was determined by phylogenesis. The results of the first study provided confirmatory data for previous studies indicating greater QA variability in HBeAg(-) than HBeAg(+) patients. The results show that high complexity in the preCore region is associated with low viral replication, in keeping with the key role of this region in HBV replication, and suggest an enhanced immune response in HBeAg(-) patients, probably related to the lack of HBeAg immunomodulatory activity. In the same direction, the positive selection of Core variants in HBeAg(-) and fluctuating status patients found in this study, can be understood as a potential mechanism to escape the host immune system by nucleocapsid sequence changes. Finally, the strong negative correlation of QA evolution in the treatment-free period and under treatment shows the importance of studying the QA before treating patients, as a potential predictive factor of HBV evolution in cases of NUC nonresponse. The results of the second study provides evidence of HBV genotype mixtures that change over time and illustrates the complex dynamics of the HBV quasispecies as an additional mechanism when adapting to new situations, such as host immune response and/or antiviral treatment. Discrepancies between genotypes in the polymerase/surface and X/preCore regions suggest phenomena of intergenotype recombination and indicate the need for a consensus effort to set an HBV genotyping approach that will lead to a more comprehensive understanding of the clinical significance of HBV genotype classification

Complejidad de la cuasiespecie del virus de la hepatitis B en la región X/preCore: asociación con la evolución de la infección con y sin tratamiento antiviral /

El Virus de la hepatitis B (VHB) tiene una tasa de mutación de 3,2×10-5 - 7,9×10-5 nucleótidos sustituidos/ciclo replicativo, esto se debe a que este ciclo consta de un paso de retrotranscripción llevado a cabo por una transcriptasa reversa que carece de actividad correctora de errores exonucleasa 3'-5'. Este hecho resulta en una enorme diversidad genética en forma de genotipos, subgenotipos, cuasiespecies (QS) y variantes en diferentes regiones del genoma. Existen numerosas evidencias de la relación entre la diversidad genética del VHB, e incluso de la complejidad de la QS, y la tasa de seroconversión HBeAg, los niveles de ADN-VHB, el escape inmune, la patogénesis y la respuesta al tratamiento, lo que pone de manifiesto la relevancia de estudiar dicha variabilidad y complejidad. El objetivo de este proyecto de tesis fue evaluar la complejidad de la QS en la región preCore/Core y su relación con el estatus HBeAg, y analizar los cambios en la QS durante la dinámica natural y bajo el tratamiento con análogos de nucleós(t)idos (NUCs), a través de los parámetros de complejidad (entropía de Shannon normalizada, frecuencia de mutación y diversidad nucleotídica). El segundo objetivo fue estudiar la complejidad a nivel de genotipos mediante la evaluación de la presencia de mezclas y sus consecuencias, en dos regiones del genoma (polimerasa/superficie y X/preCore) en pacientes con infección crónica, también durante dos períodos (dinámica natural y tratamiento con NUCs). Para ello en el primer estudio se llevó a cabo la secuenciación masiva por UDPS (ultra-deep pyrosequencing) de la región preCore/Core en muestras secuenciales (en el momento del diagnóstico, tras un tiempo sin tratamiento y después de no responder al mismo) de 10 pacientes seleccionados y agrupados según su estatus HBeAg. En el segundo estudio se analizaron muestras de otros 10 pacientes en la región polimerasa/superficie y X/preCore en una muestra basal, tras un tiempo sin tratamiento y durante el tratamiento, el genotipo se determinó por filogénesis. Los resultados del primer estudio revelan mayor complejidad de la QS en pacientes HBeAg(-) que HBeAg(+), confirmando estudios previos. Los resultados también muestran que la alta complejidad en la región preCore está asociada con una baja replicación, en concordancia con el papel clave de esta región en la misma, y sugiere una respuesta inmune aumentada en los pacientes HBeAg(-), probablemente debido a la falta de actividad inmunomoduladora de HBeAg. La selección positiva de variantes core en pacientes HBeAg o fluctuante que se ha observado, puede ser entendida como un mecanismo potencial de escape del sistema inmune por cambios en la secuencia de la nucleocápside. Por último, se ha encontrado una correlación negativa de la evolución de la QS en el período sin tratamiento y en el período con tratamiento, lo que sugiere la importancia de estudiar la complejidad de la QS antes de tratar a los pacientes como un posible factor predictivo de la evolución del virus en el caso de no respondedores a NUCs. Los resultados del segundo estudio evidencian infecciones con mezclas de genotipos y su cambio a lo largo del tiempo y ponen de manifiesto la compleja dinámica de la QS del VHB como un mecanismo adicional para adaptarse a nuevas situaciones, como pueden ser la repuesta del sistema inmune o un determinado tratamiento antiviral. Se han encontrado discrepancias entre los genotipos obtenidos en la región polimerasa/superficie y X/preCore que sugieren un fenómeno de recombinación intergenotípica y que señalan la necesidad de consensuar una región y técnica para el genotipaje, que lleve a una mejor comprensión del significado clínico de la clasificación genotípica.Hepatitis B Virus (HBV) shows a high mutation rate (3.2 × 10-5 - 7.9 × 10-5 nucleotide substitutions/replicative cycle), due to its unique life cycle which requires an error-prone reverse transcriptase for replication. This fact results in a tremendous genetic variation in the form of genotypes, sub-genotypes, quasispecies (QA) and variants in different regions of the genome. There has been considerable evidence on the relationship between HBV genetic variation, or even QA complexity, and HBeAg seroconversion, HBV-DNA levels, immune scape, pathogenesis and treatment response, showing the relevance of the analysis of this variability and complexity. The aim of this thesis project was to evaluate HBV QA complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUCs) treatment, measuring complexity parameters (normalized Shannon entropy, mutation frequency and nucleotide diversity). The second aim was to study the complexity at genotype level by assessing the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions in patients with chronic hepatitis B virus, also during two periods (natural evaluation and NUCs treatment). For this purpose ultra-deep pyrosequencing (UDPS) of HBV preCore/Core regions in sequential samples (baseline at moment of diagnosis, after a period treatment-free, and after a period of treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status, were analyzed in the first study. In the second study, thirty samples from another ten chronic hepatitis B patients were analyzed in the polymerase/surface and X/preCore regions in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment and the HBV genotype was determined by phylogenesis. The results of the first study provided confirmatory data for previous studies indicating greater QA variability in HBeAg(-) than HBeAg(+) patients. The results show that high complexity in the preCore region is associated with low viral replication, in keeping with the key role of this region in HBV replication, and suggest an enhanced immune response in HBeAg(-) patients, probably related to the lack of HBeAg immunomodulatory activity. In the same direction, the positive selection of Core variants in HBeAg(-) and fluctuating status patients found in this study, can be understood as a potential mechanism to escape the host immune system by nucleocapsid sequence changes. Finally, the strong negative correlation of QA evolution in the treatment-free period and under treatment shows the importance of studying the QA before treating patients, as a potential predictive factor of HBV evolution in cases of NUC nonresponse. The results of the second study provides evidence of HBV genotype mixtures that change over time and illustrates the complex dynamics of the HBV quasispecies as an additional mechanism when adapting to new situations, such as host immune response and/or antiviral treatment. Discrepancies between genotypes in the polymerase/surface and X/preCore regions suggest phenomena of intergenotype recombination and indicate the need for a consensus effort to set an HBV genotyping approach that will lead to a more comprehensive understanding of the clinical significance of HBV genotype classification

Significant Improvement in Diagnosis of Hepatitis C Virus Infection by a One-Step Strategy in a Central Laboratory : an Optimal Tool for Hepatitis C Elimination?

The remarkable effectivity of current antiviral therapies has led to consider the elimination of hepatitis C virus (HCV) infection. However, HCV infection is highly underdiagnosed; therefore, a global strategy for eliminating it requires improving the effectiveness of HCV diagnosis to identify hidden cases. The remarkable effectivity of current antiviral therapies has led to consider the elimination of hepatitis C virus (HCV) infection. However, HCV infection is highly underdiagnosed; therefore, a global strategy for eliminating it requires improving the effectiveness of HCV diagnosis to identify hidden cases. In this study, we assessed the effectiveness of a protocol for HCV diagnosis based on viral load reflex testing of anti-HCV antibody-positive patients (known as one-step diagnosis) by analyzing all diagnostic tests performed by a central laboratory covering an area of 1.5 million inhabitants in Barcelona, Spain, before (83,786 cases) and after (45,935 cases) the implementation of the reflex testing protocol. After its implementation, the percentage of anti-HCV-positive patients with omitted HCV RNA determination remarkably decreased in most settings, particularly in drug treatment centers and primary care settings, where omitted HCV RNA analyses had absolute reductions of 76.4 and 20.2%, respectively. In these two settings, the percentage of HCV RNA-positive patients identified as a result of reflex testing accounted for 55 and 61% of all anti-HCV-positive patients. HCV RNA results were provided in a mean of 2 days. The presence of HCV RNA and age of ≥65 years were significantly associated with advanced fibrosis, assessed using the serological FIB-4 index (odds ratio [OR], 5.92; 95% confidence interval [CI], 3.4 to 10.4). The implementation of viral load reflex testing in a central laboratory is feasible and significantly increases the diagnostic effectiveness of HCV infections, while allowing the identification of underdiagnosed cases

The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis

[Background] Respiratory failure is a key feature of severe Covid-19 and a critical driver of mortality, but for reasons poorly defined affects less than 10% of SARS-CoV-2 infected patients.[Methods] We included 1,980 patients with Covid-19 respiratory failure at seven centers in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe (Milan, Monza, Madrid, San Sebastian and Barcelona) for a genome-wide association analysis. After quality control and exclusion of population outliers, 835 patients and 1,255 population-derived controls from Italy, and 775 patients and 950 controls from Spain were included in the final analysis. In total we analyzed 8,582,968 single-nucleotide polymorphisms (SNPs) and conducted a metaanalysis of both case-control panels.[Results] We detected cross-replicating associations with rs11385942 at chromosome 3p21.31 and rs657152 at 9q34, which were genome-wide significant (P<5×10-8) in the meta-analysis of both study panels, odds ratio [OR], 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.14×10-10 and OR 1.32 (95% CI, 1.20 to 1.47; P=4.95×10-8), respectively. Among six genes at 3p21.31, SLC6A20 encodes a known interaction partner with angiotensin converting enzyme 2 (ACE2). The association signal at 9q34 was located at the ABO blood group locus and a blood-group-specific analysis showed higher risk for A-positive individuals (OR=1.45, 95% CI, 1.20 to 1.75, P=1.48×10-4) and a protective effect for blood group O (OR=0.65, 95% CI, 0.53 to 0.79, P=1.06×10-5).[Conclusions] We herein report the first robust genetic susceptibility loci for the development of respiratory failure in Covid-19. Identified variants may help guide targeted exploration of severe Covid19 pathophysiology.The IKMB's core facilities received infrastructure support by the Deutsche Forschungsgemeinschaft (DFG) Cluster of Excellence "Precision Medicine in Chronic Inflammation" (PMI, EXC2167). The project also received support through a philanthropic donation by Stein Erik Hagen and Canica AS. L.V. was funded by the Fondazione IRCCS Ca’ Granda «COVID-19 Biobank» research grant. This work was also supported by the Ministero dell’Istruzione, dell’Università e della Ricerca – MIUR project "Dipartimenti di Eccellenza 2018 – 2022" (n° D15D18000410001) to the Department of Medical Sciences, University of Torino. The IKMB authors received financial support from the UKSH Foundation "Gutes Tun!" (special thanks to Alexander Eck, Jenspeter Horst and Jens Scholz) and the German Federal Ministry of Education and Research (BMBF; grant ID 01KI20197). HLA-Typing was performed and supported by the Stefan-MorschStiftung. M.A.H was supported by the Spanish Ministry of Science and Innovation ‘JdC fellowship IJC2018-035131-I.N