Going beyond GWAS: New methods to interpret association signals

The aim of genetics is to understand the genetic basis of traits by linking genetic variability to phenotypic variability. In recent years, progress in the field of complex human trait genetics led to the discovery of thousands of common genetic variants robustly associated with complex human traits through genome-wide association studies (GWAS). However, it is currently unclear how to best to tackle the challenge of interpreting variants in the context of the biology involved. My work explored various avenues to help in this challenge. One strategy for interpretation is pathway analysis, where prior biological knowledge is formalized into sets of genes with annotated functions and results from genetics studies are searched for enrichments. Using this approach, one can connect the biological processes to the investigated trait. For this purpose, I developed a methodology to calculate pathway enrichments from GWAS results in an efficient way and in agreement with statistical principles. As a first step, the methodology combines results for SNPs in a gene region into a single gene wise p-value, with methods that are both fast and have a high level of numerical precision. The speed allows controlling the pathway enrichment step for potential correlation between genes leading to statistically correct p-values. This methodology was implemented in a software tool called Pascal. Its performance was tested on a large set of GWAS results and compared favorably to other methods. Efforts were made to ensure that the software would be easy to use by a wider community. Another challenge in the interpretation of GWAS results is to understand the reasons a genetic variant leads to changes in phenotype. Most uncovered variants seem to impact gene regulation. Therefore, understanding chromatin architecture will be crucial to understand the regulatory consequences of genetic variants. One feature of eukaryotic chromatin is that it can take the form of a compacted state making it inaccessible to most regulatory factors. To help elucidate which factors play a role in moving between compacted an open state, I developed an new method of integrative data analysis for transcription factor motif, DNase1 hypersensitivity and gene expression data. Transcription factor motif and DNase1 hypersensitivity were combined to calculate chromatin accessibility scores. These in turn were associated to gene expression using a linear mixed modeling approach. Applying this method on large public datasets predicted a set of candidate chromatin accessibility regulators. This set was heavily enriched in ’pioneer factors’: factors that can bind and open compacted chromatin, suggesting that the approach did indeed uncover regulators of chromatin accessibility. A major hindrance to the interpretation of human variants uncovered by GWAS is that it is not possible to perform genetic manipulations to validate and build on the findings. Therefore, investigations using model organisms remain relevant. To further the understanding of the genetics of fly growth control, I helped in the statistical analysis of a GWAS data set in an . The study is noteworthy for its extensive environmental control and follow-up experiments on candidate genes. -- La génétique cherche à comprendre la base génétique de caractères observables, dits phénotypes, en liant la variabilité génétique à la variabilité phénotypique. Ces dernières années, les progrès apportés à la génétique des phénotypes complexes ont amené à la découverte de milliers de variations génétiques associées significativement à des phénotypes humains complexes, au moyen de l’étude d’association pangénomique, communément appelée GWAS (de l’anglais Genome-Wide Association Study). Cependant, interpréter ces associations dans leur contexte biologique reste un défi. Mon travail a consisté à explorer différentes possibilités pour y répondre. Une des stratégies pour relier un phénotype étudié aux processus biologiques est l’analyse par voies moléculaires, où l’on recherche un enrichissement des associations GWAS parmi l’ensemble de groupes de gènes de fonctions cohérentes. Cette analyse permet ainsi de relier des processus biologiques au phénotype étudié. Dans ce but, j’ai développé une méthode calculant avec efficacité l’enrichissement des voies moléculaires des associations GWAS. La méthode combine les résultats des polymorphismes dans la région d’un gène en une probabilité pour un gène, au moyen de méthodes rapides et précises. La vitesse permet de contrôler l’étape d’enrichissement des voies pour une corrélation potentielle entre les gènes, menant à des probabilités statistiquement correctes. J’ai implémenté cette méthode dans le logiciel Pascal. Sa performance a été testé sur un large jeu de résultats GWAS et il surpasse les autres méthodes. Des efforts ont été fait afin d’assurer que le logiciel soit facile d’utilisation pour la communauté scientifique. Un autre défi lié à l’interprétation des résultats GWAS est de comprendre les raisons pour lesquelles une variation génétique résulte en un changement phénotypique. La plupart des variations découvertes semblent affecter la régulation des gènes. Ainsi, comprendre l’architecture de la chromatine est crucial pour appréhender les conséquences régulatrices de ces variations. Une des caractéristiques de la chromatine des eucaryotes est qu’elle peut être compactée, la rendant inaccessible à la plupart des facteurs de régulations. Pour trouver quels sont les facteurs jouant un rôle dans le passage entre états compacté et ouverts, j’ai développé une nouvelle méthode intégrant l’analyse des motifs de facteurs de transcription, l’hypersensibilité de la Dnase1 et les données d’expression des gènes. Les deux premiers critères ont été combiné pour calculer des scores d’accessibilité de la chromatine. Ils ont ensuite été associés à l’expression des gènes en utilisant un modèle linéaire mixte. L’application de cette méthode sur des larges données publiques a prédit des régulateurs candidats d’accessibilité de la chromatine. Ce jeu était enrichi en “facteurs pionniers”, qui s’accrochent et ouvrent la chromatine compactée, suggérant que cette approche a en effet permis de découvrir des régulateurs d’accessibilité à la chromatine. Un obstacle majeur dans l’interprétation des variations humaines découvertes par GWAS est qu’il n’est pas possible de réaliser des manipulations génétiques permettant de valider ces découvertes, d’où l’utilisation d’organismes modèles. Afin de comprendre davantage la génétique du contrôle de croissance des mouches, j’ai aidé à l’analyse statistique de données de GWAS d’une population L’étude est remarquable pour son contrôle environnemental étendu et ses expériences de suivi sur les gènes candidats

Johann J. Hagen, Die Anklagen gegen Andokides: Ein Rekonstruktionsversuch

Johann J. Hagen, Die Anklagen gegen Andokides: Ein Rekonstruktionsversuch, Hamburg, Verlag Dr. Kovač, 2021, 424 S. ISBN: 978-3-339-12522-4Johann J. Hagen, Die Anklagen gegen Andokides: Ein Rekonstruktionsversuch, Hamburg, Verlag Dr. Kovač, 2021, 424 S. ISBN: 978-3-339-12522-4Johann J. Hagen, Die Anklagen gegen Andokides: Ein Rekonstruktionsversuch, Hamburg, Verlag Dr. Kovač, 2021, 424 S. ISBN: 978-3-339-12522-

Process for recovering terephthalic acid from waste polyethylene terephthalate

Terephthalic acid (TPA) is recovered from used polyethylene terephthalate beverage containers by reacting comminuted containers in a reaction zone with an aqueous medium containing ammonium hydroxide at elevated temperatures and pressures to form a water soluble diammonium salt of TP A, separating any undissolved solids from the reaction product, acidifying the remaining reaction product to liberate TP A, and separating the precipitated TPA. A portion of the liquid remaining after separation of TPA can be mixed with lime or slaked lime, the resulting mixture treated in an ammonia stripper to remove ammonia therefrom and the ammonia overheads from the stripper blended with another portion of the liquid remaining after separation of TPA to form a stream containing ammonium hydroxide which is recycled to the reaction zone. Ethylene glycol can be recovered from the bottoms from the ammonia stripper by distillation.https://digitalcommons.mtu.edu/patents/1102/thumbnail.jp

Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases

Mapping perturbed molecular circuits that underlie complex diseases remains a great challenge. We developed a comprehensive resource of 394 cell type– and tissue-specific gene regulatory networks for human, each specifying the genome-wide connectivity among transcription factors, enhancers, promoters and genes. Integration with 37 genome-wide association studies (GWASs) showed that disease-associated genetic variants—including variants that do not reach genome-wide significance—often perturb regulatory modules that are highly specific to disease-relevant cell types or tissues. Our resource opens the door to systematic analysis of regulatory programs across hundreds of human cell types and tissue

Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.Fil: von Stetten, David. Technische Universität Berlin; AlemaniaFil: Seibeck, Sven. Freie Universität Berlin.; AlemaniaFil: Michael, Norbert. Freie Universität Berlin.; AlemaniaFil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; AlemaniaFil: Mroginski, Maria Andrea. Technische Universität Berlin; AlemaniaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; AlemaniaFil: Krauss, Norbert. Freie Universität Berlin.; AlemaniaFil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; AlemaniaFil: Hildebrandt, Peter. Technische Universität Berlin; AlemaniaFil: Borucki, Berthold. Freie Universität Berlin.; AlemaniaFil: Lamparter, Tilman. Freie Universität Berlin.; Alemani

Structural snapshot of a bacterial phytochrome in its functional intermediate state

Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors

Assessment of network module identification across complex diseases

Many bioinformatics methods have been proposed for reducing the complexity of large gene or protein networks into relevant subnetworks or modules. Yet, how such methods compare to each other in terms of their ability to identify disease-relevant modules in different types of network remains poorly understood. We launched the 'Disease Module Identification DREAM Challenge', an open competition to comprehensively assess module identification methods across diverse protein-protein interaction, signaling, gene co-expression, homology and cancer-gene networks. Predicted network modules were tested for association with complex traits and diseases using a unique collection of 180 genome-wide association studies. Our robust assessment of 75 module identification methods reveals top-performing algorithms, which recover complementary trait-associated modules. We find that most of these modules correspond to core disease-relevant pathways, which often comprise therapeutic targets. This community challenge establishes biologically interpretable benchmarks, tools and guidelines for molecular network analysis to study human disease biology

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency

Multi-layered omics approaches can help define relationships between genetic factors, biochemical processes and phenotypes thus extending research of inherited diseases beyond identifying their monogenic cause 1. We implemented a multi-layered omics approach for the inherited metabolic disorder methylmalonic aciduria (MMA). We performed whole genome sequencing, transcriptomic sequencing, and mass spectrometry-based proteotyping from matched primary fibroblast samples of 230 individuals (210 affected, 20 controls) and related the molecular data to 105 phenotypic features. Integrative analysis identified a molecular diagnosis for 84% (177/210) of affected individuals, the majority (148) of whom had pathogenic variants in methylmalonyl-CoA mutase (MMUT). Untargeted analysis of all three omics layers revealed dysregulation of the TCA cycle and surrounding metabolic pathways, a finding that was further corroborated by multi-organ metabolomics of a hemizygous Mmut mouse model. Integration of phenotypic disease severity indicated downregulation of oxoglutarate dehydrogenase and upregulation of glutamate dehydrogenase, two proteins involved in glutamine anaplerosis of the TCA cycle. The relevance of disturbances in this pathway was supported by metabolomics and isotope tracing studies which showed decreased glutamine-derived anaplerosis in MMA. We further identified MMUT to physically interact with both, oxoglutarate dehydrogenase complex components and glutamate dehydrogenase providing evidence for a multi-protein metabolon that orchestrates TCA cycle anaplerosis. This study emphasizes the utility of a multi-modal omics approach to investigate metabolic diseases and highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA. Take home message Combination of integrative multi-omics technologies with clinical and biochemical features leads to an increased diagnostic rate compared to genome sequencing alone and identifies anaplerotic rewiring as a targetable feature of the rare inborn error of metabolism methylmalonic aciduria

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae