Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications

GCAT|Panel, a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies

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.publishedVersio

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

Integrative analysis of the functional consequences of inversions in the human genome

La variación estructural contribuye de forma substancial a la diversidad genética, pero su asociación con rasgos complejos y enfermedades no se entiende del todo y merece una caracterización detallada. Esto es especialmente cierto para las inversiones, cuyas consecuencias funcionales han permanecido ocultas, con algunas pocas excepciones. A pesar del creciente interés en caracterizar todo tipo de variantes genómicas, las inversiones se han dejado a menudo de lado debido a la presencia de regiones repetitivas en sus puntos de rotura sumado a su naturaleza balanceada. El proyecto InvFEST ha tratado de superar este desafío técnico mediante el desarrollo de nuevos métodos para genotipar inversiones. Gracias a este esfuerzo, un total de 111 inversiones comunes en humanos han sido genotipadas con precisión en un gran número de individuos de diversas poblaciones, convirtiéndose en el recurso más completo y fiable de este tipo de variación disponible hasta la fecha. En la era actual de la medicina de precisión, el análisis de loci de carácteres cuantitativos (QTL) ha surgido como un enfoque clave para determinar cómo los polimorfismos genéticos influyen en la expresión génica y, a su vez, en los rasgos fenotípicos. Por lo tanto, esta tesis aprovecha al máximo los datos generados para realizar por primera vez una cuantificación sistemática del impacto funcional de las inversiones polimórficas humanas. Los resultados muestran que las inversiones pueden afectar la expresión génica ya sea manteniendo haplotipos diferenciados, alterando o reoganizando la estructura de los genes, creando nuevos transcritos de fusión, o actuando a través de cambios en los patrones epigenéticos. Sorprendentemente, la mitad de las inversiones analizadas actúan como QTL principales o están en un desequilibrio de ligamiento (LD) con QTL centinelas para la expresión génica y los cambios epigenéticos en diferentes tejidos y líneas celulares, lo que sugiere que las inversiones están enriquecidas en efectos funcionales. En particular, esta influencia sobre fenotipos moleculares es aún más acusada para las inversiones largas (>100 kb), ya que están involucradas en el 80% de los QTL identificados. Aunque se sabe que las variantes estructurales tienen una mayor probabilidad de estar asociadas con niveles de expresión y rasgos complejos, los efectos detectados para las inversiones podrían reflejar un compromiso entre cambios de expresión beneficiosos y el posible impacto negativo sobre la fertilidad debido a la producción de gametos desequilibrados por recombinación. Además, las regiones de las inversiones presentan un enriquecimiento de señales de estudios de asociación a nivel de todo el genoma, y 14 de ellas están en alto LD con variantes asociadas a diferentes caracteres, lo que apoya su posible implicación en fenotipos humanos. Finalmente, se han investigado en detalle dos inversiones interesantes. HsInv0102 invierte un exón alternativo no codificante del gen RHOH. Curiosamente, esta inversión también está asociada con los niveles de la proteína RhoH y se ha estimado que el alelo invertido actúa como un locus protector con efectos moderados sobre la susceptibilidad al cáncer sanguíneo. Por otra parte, HsInv0124 regula la expresión de varios genes en la región IFITM, como IFITM2 e IFITM3, a través de cambios en los patrones de modificación de histonas. Además, bajo condiciones de infección, esta inversión tiene un efecto más generalizado sobre la expresión de genes relacionados con la respuesta inmune, lo que indica que puede desempeñar un papel importante en la defensa contra las infecciones víricas. En su conjunto, estos hallazgos ilustran el posible impacto funcional de las inversiones en el genoma humano y ayudan a desentrañar la relación que existe entre estas variantes y la variabilidad fenotípica.Structural variation contributes substantially to the genetic diversity, but its association with complex traits and diseases is not well understood and deserves detailed characterisation. This is particularly true for chromosomal inversions, whose functional consequences have remained elusive in humans, with very few notable exceptions. Despite the rising interest in identifying all types of genomic variants, inversions have been often set aside due to the presence of repetitive regions in their breakpoints along with their balanced nature. The InvFEST Project has tried to overcome this technical challenge by developing unique methods for inversion genotyping. Thanks to this effort, a total of 111 polymorphic human inversions have been accurately genotyped in a large number of individuals from diverse populations, becoming the most complete and reliable resource of this type of variation available to date. In the current era of precision medicine, quantitative trait loci (QTL) analysis has emerged as a key approach to determine how genetic polymorphisms influence gene expression and, in turn, phenotypic traits. Thus, this thesis makes the most of the great amount of data generated to perform for the first time a systematic quantification of the functional impact of human polymorphic inversions. The results show that inversions can affect gene expression by maintaining differentiated haplotypes, disrupting or reorganizing gene structures, creating novel fusion transcripts or acting through changes in epigenetic patterns. Strikingly, half of the inversions analysed act as lead QTLs or are in high linkage disequilibrium (LD) with top QTLs for gene expression and epigenetic changes across different tissues and cell lines, which suggests that inversions are enriched for functional effects. In particular, this influence on molecular phenotypes is even stronger for long inversions (>100kb), which are involved in 80% of the QTL associations. Although structural variants are known to have a higher chance to be associated with expression levels and complex traits, the detected inversion effects may reflect a trade-off between beneficial expression changes and potential negative costs on fertility due to production of unbalanced gametes by recombination. Furthermore, inversions present an enrichment of genome-wide association studies (GWAS) signals in their surrounding area, and 14 of them are in high LD with trait-associated variants, supporting their potential implication in human phenotypes. Finally, the phenotypic consequences of two interesting inversions have been investigated in detail. HsInv0102 inverts an alternative non-coding exon from the RHOH gene. Interestingly, this inversion is also associated with RhoH protein levels and the inverted allele could act as a moderate protective locus on blood cancer susceptibility. On the other hand, HsInv0124 regulates the expression of several genes in the IFITM region, including IFITM2 and IFITM3, through changes in histone modification patterns. Moreover, under infection conditions, this inversion has a pervasive effect on the expression of genes related with immune response, indicating that it may play an important role in defense against viral infections. All together, these findings illustrate the potential functional impact of inversions on the human genome and help to uncover previously missing variants related to phenotype variability

Integrative analysis of the functional consequences of inversions in the human genome

La variación estructural contribuye de forma substancial a la diversidad genética, pero su asociación con rasgos complejos y enfermedades no se entiende del todo y merece una caracterización detallada. Esto es especialmente cierto para las inversiones, cuyas consecuencias funcionales han permanecido ocultas, con algunas pocas excepciones. A pesar del creciente interés en caracterizar todo tipo de variantes genómicas, las inversiones se han dejado a menudo de lado debido a la presencia de regiones repetitivas en sus puntos de rotura sumado a su naturaleza balanceada. El proyecto InvFEST ha tratado de superar este desafío técnico mediante el desarrollo de nuevos métodos para genotipar inversiones. Gracias a este esfuerzo, un total de 111 inversiones comunes en humanos han sido genotipadas con precisión en un gran número de individuos de diversas poblaciones, convirtiéndose en el recurso más completo y fiable de este tipo de variación disponible hasta la fecha. En la era actual de la medicina de precisión, el análisis de loci de carácteres cuantitativos (QTL) ha surgido como un enfoque clave para determinar cómo los polimorfismos genéticos influyen en la expresión génica y, a su vez, en los rasgos fenotípicos. Por lo tanto, esta tesis aprovecha al máximo los datos generados para realizar por primera vez una cuantificación sistemática del impacto funcional de las inversiones polimórficas humanas. Los resultados muestran que las inversiones pueden afectar la expresión génica ya sea manteniendo haplotipos diferenciados, alterando o reoganizando la estructura de los genes, creando nuevos transcritos de fusión, o actuando a través de cambios en los patrones epigenéticos. Sorprendentemente, la mitad de las inversiones analizadas actúan como QTL principales o están en un desequilibrio de ligamiento (LD) con QTL centinelas para la expresión génica y los cambios epigenéticos en diferentes tejidos y líneas celulares, lo que sugiere que las inversiones están enriquecidas en efectos funcionales. En particular, esta influencia sobre fenotipos moleculares es aún más acusada para las inversiones largas (>100 kb), ya que están involucradas en el 80% de los QTL identificados. Aunque se sabe que las variantes estructurales tienen una mayor probabilidad de estar asociadas con niveles de expresión y rasgos complejos, los efectos detectados para las inversiones podrían reflejar un compromiso entre cambios de expresión beneficiosos y el posible impacto negativo sobre la fertilidad debido a la producción de gametos desequilibrados por recombinación. Además, las regiones de las inversiones presentan un enriquecimiento de señales de estudios de asociación a nivel de todo el genoma, y 14 de ellas están en alto LD con variantes asociadas a diferentes caracteres, lo que apoya su posible implicación en fenotipos humanos. Finalmente, se han investigado en detalle dos inversiones interesantes. HsInv0102 invierte un exón alternativo no codificante del gen RHOH. Curiosamente, esta inversión también está asociada con los niveles de la proteína RhoH y se ha estimado que el alelo invertido actúa como un locus protector con efectos moderados sobre la susceptibilidad al cáncer sanguíneo. Por otra parte, HsInv0124 regula la expresión de varios genes en la región IFITM, como IFITM2 e IFITM3, a través de cambios en los patrones de modificación de histonas. Además, bajo condiciones de infección, esta inversión tiene un efecto más generalizado sobre la expresión de genes relacionados con la respuesta inmune, lo que indica que puede desempeñar un papel importante en la defensa contra las infecciones víricas. En su conjunto, estos hallazgos ilustran el posible impacto funcional de las inversiones en el genoma humano y ayudan a desentrañar la relación que existe entre estas variantes y la variabilidad fenotípica.Structural variation contributes substantially to the genetic diversity, but its association with complex traits and diseases is not well understood and deserves detailed characterisation. This is particularly true for chromosomal inversions, whose functional consequences have remained elusive in humans, with very few notable exceptions. Despite the rising interest in identifying all types of genomic variants, inversions have been often set aside due to the presence of repetitive regions in their breakpoints along with their balanced nature. The InvFEST Project has tried to overcome this technical challenge by developing unique methods for inversion genotyping. Thanks to this effort, a total of 111 polymorphic human inversions have been accurately genotyped in a large number of individuals from diverse populations, becoming the most complete and reliable resource of this type of variation available to date. In the current era of precision medicine, quantitative trait loci (QTL) analysis has emerged as a key approach to determine how genetic polymorphisms influence gene expression and, in turn, phenotypic traits. Thus, this thesis makes the most of the great amount of data generated to perform for the first time a systematic quantification of the functional impact of human polymorphic inversions. The results show that inversions can affect gene expression by maintaining differentiated haplotypes, disrupting or reorganizing gene structures, creating novel fusion transcripts or acting through changes in epigenetic patterns. Strikingly, half of the inversions analysed act as lead QTLs or are in high linkage disequilibrium (LD) with top QTLs for gene expression and epigenetic changes across different tissues and cell lines, which suggests that inversions are enriched for functional effects. In particular, this influence on molecular phenotypes is even stronger for long inversions (>100kb), which are involved in 80% of the QTL associations. Although structural variants are known to have a higher chance to be associated with expression levels and complex traits, the detected inversion effects may reflect a trade-off between beneficial expression changes and potential negative costs on fertility due to production of unbalanced gametes by recombination. Furthermore, inversions present an enrichment of genome-wide association studies (GWAS) signals in their surrounding area, and 14 of them are in high LD with trait-associated variants, supporting their potential implication in human phenotypes. Finally, the phenotypic consequences of two interesting inversions have been investigated in detail. HsInv0102 inverts an alternative non-coding exon from the RHOH gene. Interestingly, this inversion is also associated with RhoH protein levels and the inverted allele could act as a moderate protective locus on blood cancer susceptibility. On the other hand, HsInv0124 regulates the expression of several genes in the IFITM region, including IFITM2 and IFITM3, through changes in histone modification patterns. Moreover, under infection conditions, this inversion has a pervasive effect on the expression of genes related with immune response, indicating that it may play an important role in defense against viral infections. All together, these findings illustrate the potential functional impact of inversions on the human genome and help to uncover previously missing variants related to phenotype variability

Integrative analysis of the functional consequences of inversions in the human genome /

Departament responsable de la tesi: Departament de Genètica i de Microbiologia.La variación estructural contribuye de forma substancial a la diversidad genética, pero su asociación con rasgos complejos y enfermedades no se entiende del todo y merece una caracterización detallada. Esto es especialmente cierto para las inversiones, cuyas consecuencias funcionales han permanecido ocultas, con algunas pocas excepciones. A pesar del creciente interés en caracterizar todo tipo de variantes genómicas, las inversiones se han dejado a menudo de lado debido a la presencia de regiones repetitivas en sus puntos de rotura sumado a su naturaleza balanceada. El proyecto InvFEST ha tratado de superar este desafío técnico mediante el desarrollo de nuevos métodos para genotipar inversiones. Gracias a este esfuerzo, un total de 111 inversiones comunes en humanos han sido genotipadas con precisión en un gran número de individuos de diversas poblaciones, convirtiéndose en el recurso más completo y fiable de este tipo de variación disponible hasta la fecha. En la era actual de la medicina de precisión, el análisis de loci de carácteres cuantitativos (QTL) ha surgido como un enfoque clave para determinar cómo los polimorfismos genéticos influyen en la expresión génica y, a su vez, en los rasgos fenotípicos. Por lo tanto, esta tesis aprovecha al máximo los datos generados para realizar por primera vez una cuantificación sistemática del impacto funcional de las inversiones polimórficas humanas. Los resultados muestran que las inversiones pueden afectar la expresión génica ya sea manteniendo haplotipos diferenciados, alterando o reoganizando la estructura de los genes, creando nuevos transcritos de fusión, o actuando a través de cambios en los patrones epigenéticos. Sorprendentemente, la mitad de las inversiones analizadas actúan como QTL principales o están en un desequilibrio de ligamiento (LD) con QTL centinelas para la expresión génica y los cambios epigenéticos en diferentes tejidos y líneas celulares, lo que sugiere que las inversiones están enriquecidas en efectos funcionales. En particular, esta influencia sobre fenotipos moleculares es aún más acusada para las inversiones largas (>100 kb), ya que están involucradas en el 80% de los QTL identificados. Aunque se sabe que las variantes estructurales tienen una mayor probabilidad de estar asociadas con niveles de expresión y rasgos complejos, los efectos detectados para las inversiones podrían reflejar un compromiso entre cambios de expresión beneficiosos y el posible impacto negativo sobre la fertilidad debido a la producción de gametos desequilibrados por recombinación. Además, las regiones de las inversiones presentan un enriquecimiento de señales de estudios de asociación a nivel de todo el genoma, y 14 de ellas están en alto LD con variantes asociadas a diferentes caracteres, lo que apoya su posible implicación en fenotipos humanos. Finalmente, se han investigado en detalle dos inversiones interesantes. HsInv0102 invierte un exón alternativo no codificante del gen RHOH. Curiosamente, esta inversión también está asociada con los niveles de la proteína RhoH y se ha estimado que el alelo invertido actúa como un locus protector con efectos moderados sobre la susceptibilidad al cáncer sanguíneo. Por otra parte, HsInv0124 regula la expresión de varios genes en la región IFITM, como IFITM2 e IFITM3, a través de cambios en los patrones de modificación de histonas. Además, bajo condiciones de infección, esta inversión tiene un efecto más generalizado sobre la expresión de genes relacionados con la respuesta inmune, lo que indica que puede desempeñar un papel importante en la defensa contra las infecciones víricas. En su conjunto, estos hallazgos ilustran el posible impacto funcional de las inversiones en el genoma humano y ayudan a desentrañar la relación que existe entre estas variantes y la variabilidad fenotípica.Structural variation contributes substantially to the genetic diversity, but its association with complex traits and diseases is not well understood and deserves detailed characterisation. This is particularly true for chromosomal inversions, whose functional consequences have remained elusive in humans, with very few notable exceptions. Despite the rising interest in identifying all types of genomic variants, inversions have been often set aside due to the presence of repetitive regions in their breakpoints along with their balanced nature. The InvFEST Project has tried to overcome this technical challenge by developing unique methods for inversion genotyping. Thanks to this effort, a total of 111 polymorphic human inversions have been accurately genotyped in a large number of individuals from diverse populations, becoming the most complete and reliable resource of this type of variation available to date. In the current era of precision medicine, quantitative trait loci (QTL) analysis has emerged as a key approach to determine how genetic polymorphisms influence gene expression and, in turn, phenotypic traits. Thus, this thesis makes the most of the great amount of data generated to perform for the first time a systematic quantification of the functional impact of human polymorphic inversions. The results show that inversions can affect gene expression by maintaining differentiated haplotypes, disrupting or reorganizing gene structures, creating novel fusion transcripts or acting through changes in epigenetic patterns. Strikingly, half of the inversions analysed act as lead QTLs or are in high linkage disequilibrium (LD) with top QTLs for gene expression and epigenetic changes across different tissues and cell lines, which suggests that inversions are enriched for functional effects. In particular, this influence on molecular phenotypes is even stronger for long inversions (>100kb), which are involved in 80% of the QTL associations. Although structural variants are known to have a higher chance to be associated with expression levels and complex traits, the detected inversion effects may reflect a trade-off between beneficial expression changes and potential negative costs on fertility due to production of unbalanced gametes by recombination. Furthermore, inversions present an enrichment of genome-wide association studies (GWAS) signals in their surrounding area, and 14 of them are in high LD with trait-associated variants, supporting their potential implication in human phenotypes. Finally, the phenotypic consequences of two interesting inversions have been investigated in detail. HsInv0102 inverts an alternative non-coding exon from the RHOH gene. Interestingly, this inversion is also associated with RhoH protein levels and the inverted allele could act as a moderate protective locus on blood cancer susceptibility. On the other hand, HsInv0124 regulates the expression of several genes in the IFITM region, including IFITM2 and IFITM3, through changes in histone modification patterns. Moreover, under infection conditions, this inversion has a pervasive effect on the expression of genes related with immune response, indicating that it may play an important role in defense against viral infections. All together, these findings illustrate the potential functional impact of inversions on the human genome and help to uncover previously missing variants related to phenotype variability

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

Metabolic associated fatty liver disease (MAFLD) is the most prevalent form of liver disease worldwide, accounting for a high liver-related mortality and morbidity with extensive multi organ involvement. This entity has displaced viral hepatitis as the main cause of severe forms of hepatic diseases, although the onset and transition of MAFLD stages still remains unclear. Nevertheless, innate and adaptive immune responses seem to play an essential role in the establishment and further progression of this disease. The immune system is responsible of safeguard and preserves organs and systems function, and might be altered under different stimuli. Thus, the liver suffers from metabolic and immune changes leading to different injuries and loss of function. It has been stablished that cell-cell crosstalk is a key process in the hepatic homeostasis maintenance. There is mounting evidence suggesting that MAFLD pathogenesis is determined by a complex interaction of environmental, genetic and host factors that leads to a full plethora of outcomes. Therefore, herein we will revisit and discuss the interplay between immune mechanisms and MAFLD, highlighting the potential role of immunological markers in an attempt to clarify its relationship.Consejería de Salud , Junta de AndalucíaMinisterio de Economía y Competitividad, Gobierno de Españ

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications