Functional analysis of genetic variants: contribution to the diagnosis of inherited metabolic diseases

Functional analysis of variants of unknown significance is gaining more importance in the development of a sound diagnostic strategy. The abundance of sequencing methods changed the molecular genetics in the field of inborn errors of metabolism. The large amount of data opens more opportunities for diagnostics but ironically, also brings more challenges in differentiating pathogenic variants from the non-pathogenic ones. Historically, to establish a biochemical diagnosis, functional diagnostic tests (like metabolite screening and enzyme assays) are used as first tier. Subsequently, targeted Sanger sequencing is employed for genetic diagnosis. Over the years, various DNA sequencing techniques are being more and more used at earlier stages in the diagnostic work up. As most of the time these investigations do not directly result in a final diagnosis, additional approaches are needed for variant classification. A great array of computational tools are currently used for the interpretation of pathogenicity of variants. However, their predictions are often in disagreement, which makes it difficult to decide which one is the most accurate. In contrast to the computational tools, functional assays have the potential to confirm or rule out variant pathogenicity. When functional tests demonstrate the pathogenicity of novel variants, in most cases, the diagnosis is confirmed. However, if the studied variants prove to be non-pathogenic, it creates awareness that further studies are necessary. This is particularly relevant for variants identified via WES/WGS in patients with a broad clinical phenotype or with a phenotype that appears to fit with the presumed pathogenic variants/genes. There is a large choice of functional studies that can be used, from targeted to untargeted approaches. The translational research presented in this thesis, has implications in patient care and is already (partly) implemented in diagnostics in our laboratory. Our work focuses on functional characterization of missense variants in genes involved in 2-hydroxyglutaric acidurias (SLC25A1 - chapter 2 and 3, and D2HGDH - chapter 4), in GABA metabolism (ALDH5A1 - chapter 5 and 6) and cerebral creatine deficiency syndromes (SLC6A8 - chapter 7). All these disorders are rare inborn errors of metabolism, for which the pathophysiology is not completely elucidated and for which no treatment is available. Our studies extended the molecular genetics of the addressed metabolic disorders, by identification of the gene associated with the D/L-2-HGA (chapter 2) and by identification of novel variants in other deficiencies (chapter 3, 4, 5, 6 and 7). Accordingly, the functional assessment of missense variants in these genes and their clinical implications are discussed in the mentioned chapters. One of the most used functional analysis technique in VUS research, is transient transfections of recombinant genes into cell models. In our experience, optimal expression of the proteins of interest can be achieved in a relatively short time (one to three days), facilitating a rapid diagnosis, especially important when time is crucial (e.g. prenatal diagnosis). Alternatively, more advanced gene editing strategies can be used. In chapter 5, we used the Flp-In system in corroboration with CRISPR Cas9 knock out technique to generate SSADH deficient HEK293-Flp-In cell lines, in which a select number of ALDH5A1 missense variants were stably expressed. The majority of the variants addressed in this thesis resulted in impaired activity, indicating that the investigated amino acid residues are essential for proper protein function. Still, the classification of missense variants with high residual activity can remain challenging. It is important to realise that the more aspects are taken into account, the more accurate the variant classification and ultimately the patient diagnosis will be. Functional data, in collaboration with broad evaluation of clinical, biochemical, in silico and genetic data, are the desired combinatorial approach for a correct diagnosis

Eigenvalues of Non-Backtracking Walks in a Cycle with Random Loops

In this paper we take a very special model of a random non-regular graph and study its non-backtracking spectrum. We study graphs consisting of a cycle with some random loops added; the graphs are not regular and their non-backtracking spectrum does not seem to be confined to some one-dimensional set in the complex plane. The non-backtracking spectrum is required in some applications, and has no straightforward connection to the usual adjacency matrix spectrum for general graphs, unlike the situation for regular graphs. Experimentally, the random graphs\u27 spectrum appears similar in shape to its deterministic counterpart, but differs because the eigenvalues are visibly clustered, especially with a mysterious gap around Re(l)=1

Galaxies with Shells in the Illustris Simulation: Metallicity Signatures

Stellar shells are low surface brightness arcs of overdense stellar regions, extending to large galactocentric distances. In a companion study, we identified 39 shell galaxies in a sample of 220 massive ellipticals ($\mathrm{M}_{\mathrm{200crit}}>6\times10^{12}\,\mathrm{M}_\odot$) from the Illustris cosmological simulation. We used stellar history catalogs to trace the history of each individual star particle inside the shell substructures, and we found that shells in high-mass galaxies form through mergers with massive satellites (stellar mass ratios $\mu_{\mathrm{stars}}\gtrsim1:10$). Using the same sample of shell galaxies, the current study extends the stellar history catalogs in order to investigate the metallicity of stellar shells around massive galaxies. Our results indicate that outer shells are often times more metal-rich than the surrounding stellar material in a galaxy's halo. For a galaxy with two different satellites forming $z=0$ shells, we find a significant difference in the metallicity of the shells produced by each progenitor. We also find that shell galaxies have higher mass-weighted logarithmic metallicities ([Z/H]) at $2$-$4\,\mathrm{R}_{\mathrm{eff}}$ compared to galaxies without shells. Our results indicate that observations comparing the metallicities of stars in tidal features, such as shells, to the average metallicities in the stellar halo can provide information about the assembly histories of galaxies.Comment: 15 pages, 5 figures. Article published in a special issue of MDPI Galaxies after the conference "On the Origin (and Evolution) of Baryonic Galaxy Halos", Galapagos Islands, 201

Formation and Incidence of Shell Galaxies in the Illustris Simulation

Shells are low surface brightness tidal debris that appear as interleaved caustics with large opening angles, often situated on both sides of the galaxy center. In this paper, we study the incidence and formation processes of shell galaxies in the cosmological gravity+hydrodynamics Illustris simulation. We identify shells at redshift z=0 using stellar surface density maps, and we use stellar history catalogs to trace the birth, trajectory and progenitors of each individual star particle contributing to the tidal feature. Out of a sample of the 220 most massive galaxies in Illustris ($\mathrm{M}_{\mathrm{200crit}}>6\times10^{12}\,\mathrm{M}_{\odot}$), $18\%\pm3\%$ of the galaxies exhibit shells. This fraction increases with increasing mass cut: higher mass galaxies are more likely to have stellar shells. Furthermore, the fraction of massive galaxies that exhibit shells decreases with increasing redshift. We find that shell galaxies observed at redshift $z=0$ form preferentially through relatively major mergers ($\gtrsim$1:10 in stellar mass ratio). Progenitors are accreted on low angular momentum orbits, in a preferred time-window between $\sim$4 and 8 Gyrs ago. Our study indicates that, due to dynamical friction, more massive satellites are allowed to probe a wider range of impact parameters at accretion time, while small companions need almost purely radial infall trajectories in order to produce shells. We also find a number of special cases, as a consequence of the additional complexity introduced by the cosmological setting. These include galaxies with multiple shell-forming progenitors, satellite-of-satellites also forming shells, or satellites that fail to produce shells due to multiple major mergers happening in quick succession.Comment: 27 pages, 18 figures. Accepted for publication in MNRAS (new figures 3 and D1 + additional minor changes to match accepted version

Formation and Incidence of Shell Galaxies in the Illustris Simulation

Shells are low surface brightness tidal debris that appear as interleaved caustics with large opening angles, often situated on both sides of the galaxy center. In this paper, we study the incidence and formation processes of shell galaxies in the cosmological gravity+hydrodynamics Illustris simulation. We identify shells at redshift z=0 using stellar surface density maps, and we use stellar history catalogs to trace the birth, trajectory and progenitors of each individual star particle contributing to the tidal feature. Out of a sample of the 220 most massive galaxies in Illustris ($\mathrm{M}_{\mathrm{200crit}}>6\times10^{12}\,\mathrm{M}_{\odot}$), $18\%\pm3\%$ of the galaxies exhibit shells. This fraction increases with increasing mass cut: higher mass galaxies are more likely to have stellar shells. Furthermore, the fraction of massive galaxies that exhibit shells decreases with increasing redshift. We find that shell galaxies observed at redshift $z=0$ form preferentially through relatively major mergers ($\gtrsim$1:10 in stellar mass ratio). Progenitors are accreted on low angular momentum orbits, in a preferred time-window between $\sim$4 and 8 Gyrs ago. Our study indicates that, due to dynamical friction, more massive satellites are allowed to probe a wider range of impact parameters at accretion time, while small companions need almost purely radial infall trajectories in order to produce shells. We also find a number of special cases, as a consequence of the additional complexity introduced by the cosmological setting. These include galaxies with multiple shell-forming progenitors, satellite-of-satellites also forming shells, or satellites that fail to produce shells due to multiple major mergers happening in quick succession.Comment: 27 pages, 18 figures. Accepted for publication in MNRAS (new figures 3 and D1 + additional minor changes to match accepted version

I Am Dissolving into Categories and Labels - Agency Affordances for Embedding and Practicing Digital Sovereignty

While the notion of digital sovereignty is loaded with a multitude of meanings referring to various actors, values and contexts, this paper is interested in how to actualize individual digital sovereignty. We do so by introducing the concept of agency affordances, which we see as a precondition for achieving digital sovereignty. We understand this notion as the ability to exercise power to, as autonomy and agency for (digital) self-sovereignty, and as power over the infrastructural sovereignty of the privately owned automated decision-making systems (ADM) systems of digital media platforms. Building our characterization of digital sovereignty on an empirical inquiry into individuals' requirements for agency, our analysis shows that digital sovereignty consists of two distinct but interrelated elements - data sovereignty and algorithmic sovereignty. Enabling practicable digital sovereignty through agency affordances, however, will require going beyond the just technical and extending towards the wider societal (infra)structures. We outline some initial steps on how to achieve that