The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

The Microsporidia are obligate intracellular parasites that have jettisoned oxidation phosphorylative capabilities during their early evolutionary history and so rely on ATP import from their host and glycolysis for their energy needs. Some species form tight associations with the host’s mitochondria and this is thought to facilitate ATP sequestration by the developing intracellular microsporidian. The human parasite, Enterocytozoon bieneusi has however lost glycolytic capabilities and may rely entirely on ATP import from its host for energy. E. bieneusi belongs to the Enterocytozoonidae microsporidian family and recent rDNA-based phylogenetic studies have suggested it has close evolutionary ties with Enterospora canceri, a crab-infecting intranuclear parasite. Such a close evolutionary relationship implied that glycolysis might also be absent in the intranuclear parasite raising questions as to how this parasite obtains energy from its unusual niche that is physically walled off from the host mitochondria, the main source of ATP in the host cell. In this study, draft genomes of four species of the Enterocytozoonidae namely, Ent. canceri, E. hepatopenaei, Hepatospora eriocheir and Hepatospora eriocheir canceri and one non-Enterocytozoonidae species, Thelohania sp. were assembled and annotated (The genome assembly of Hepatospora eriocheir was provided by Dr. Bryony Williams). Phylogenomics performed with this and publicly available genomic data confirmed the close evolutionary ties between Ent. canceri and E. bieneusi. Comparative genomic analyses also revealed that glycolysis is indeed lost in all members of the Enterocytozoonidae family sequenced in this study, hinting to the relaxation of evolutionary pressures to maintain this pathway at the base of this microsporidian family. Despite this absence, the hexokinase gene was retained in all aglycolytic genomes analysed, and that of Ent. canceri was fused to a PTPA gene. Functional assays and yeast complementation assays suggest that this chimera is able to recognise glucose as a substrate but the heterologously expressed homolog of H. eriocheir cannot. Finally, phylogenomics have been used here to demonstrate that despite the morphological differences between three Hepatospora-like organisms parasitizing different crab hosts, they are the same species. This finding adds more weight to current evidence suggesting that morphology is not an ideal marker for taxonomical classification in the Microsporidia.

Escherichia coli est une espèce commensale du tube digestif, mais elle peut aussi se révéler être un dangereux pathogène intra ou extra intestinal. Un même clone pouvant passer d'un état commensal à pathogène, la compréhension des mécanismes impliqués dans la diversification d'E. coli dans ces deux habitats représente un enjeu majeur de santé publique. Des expériences d'évolution expérimentale utilisant E. coli ont permis de révéler différentes facettes de l'adaptation bactérienne. Cependant, ces expériences de laboratoire utilisant des conditions artificielles, on peut s'interroger sur la pertinence des observations qui en découlent en milieu naturel et plus globalement s’interroger sur la part de la sélection naturelle dans la diversification de E. coli dans la nature. Pour répondre à ces questions, j'ai analysé les profils génomiques de diversification de E. coli au cours (1) d’une adaptation au tube digestif de souris ou (2) dans des infections extra-intestinales. Dans les deux cas, j’ai pu montrer une importante convergence au niveau du gène : un même gène étant muté plusieurs fois indépendamment, un signe que l’adaptation est active. Dans les infections aigues, des mutations touchant des régulateurs globaux ont été retrouvées, alors que dans le tube digestif les cibles de l’adaptation semblaient plus spécifiques. Enfin, les échantillons issus des infections incluant des souches a fort taux de mutation dites mutatrices, j'ai pu documenter pour la première fois la génomique de l'émergence de bactéries mutatrices en milieu naturel.En conclusion, mes travaux montrent que l’adaptation joue un rôle important dans la diversification de E. coli en milieu naturel et que ce processus s’apparente à celui observé dans des milieux artificiels de laboratoire. L’adaptation semble néanmoins plus active en conditions d’infections aigues que dans le tube digestif de souris. / Escherichia coli is a commensal species living in the digestive tract of vertebrates, but can also be a harmful pathogen involved in both intra and extraintestinal diseases. As clones can behave both as commensals and pathogens, the comprehension of the mechanisms involved in the diversification of E. coli in those two habitats represents a major public health concern. In vitro experimental evolution studies using E. coli have unraveled the different faces of bacterial adaptation. However, as those experiments used artificial conditions, the relevance of these observations and more generally the contribution of adaptation to the diversification of E. coli in the wild remain questionable. To answer these questions, I analyzed the genomic profiles of diversification of E. coli during (1) adaptation to the mice digestive tract or (2) during acute extraintestinal infections. In both cases, I found a strong convergence at the gene level, i.e. observation of several impendent mutations in the same gene, suggesting a dynamic adaptation. In acute infections, mutations in global regulators were recovered, while more specific genes were recruited in the mice gut. Finally, the existence of clones with high mutation rate in the infections, allowed me to document for the first time the genomics of mutator emergence in the wild. In conclusion, my work shows that adaptation is playing an important role in the diversification of E. coli, and that this process is fairly similar to the one observed in the laboratory. Nevertheless, adaptation seems more active during infections than in the mice gut.

L'endocardite infectieuse (EI) est une complication rare mais gravissime de la bactériémie à Staphylococcus aureus. Bien que certains facteurs de risque liés à l'hôte aient été décrits, l'implication de facteurs bactériens dans la survenue de l'EI est encore inconnue. Ces travaux de thèse ont visé à chercher tout élément bactérien associé à l'EI. Les facteurs phénotypiques décrits ou supposés comme potentiellement impliqués dans l'EI ont été testés. En parallèle, les profils génotypiques des souches obtenus par puces ADN ont été analysés par différents outils statistiques. L'analyse statistique univariée n'a montré aucune différence significative entre souches d'EI et souches de bactériémie, suggérant un processus complexe et multifactoriel. En effet, l'analyse discriminante en composante principale appliquée sur les données de puces ADN a permis de mettre en évidence une distinction entre les deux groupes de souches, confirmée sur une collection indépendante de souches. De plus, une fonction linéaire simplifiée, basée sur seulement 8 marqueurs génétiques, a permis d'obtenir des performances similaires, sur la collection de souches initiale ainsi que la collection indépendante de validation. En dernier lieu, les souches d'EI et de bactériémie ont été comparées à partir de séquences du génome complet (n = 40 (20 EI, 20 bactériémies)). L'analyse statistique par analyse discriminante en composante principale réalisée sur ces données génomiques confirme une distinction possible entre les deux groupes de souches. Au total, ces travaux de thèse apportent la preuve de concept que les facteurs bactériens sont impliqués dans la survenue de l'EI au cours de bactériémie à S. aureus / Infective endocarditis (IE) is a severe condition complicating 10-25% of Staphylococcus aureus bacteremia. Although host-related IE risk factors have been identified, the involvement of bacterial features in IE complication is still unclear. This PhD work aimed to characterize strictly defined IE and bacteremia isolates and searched for discriminant features. Phenotypic traits previously reported or hypothesized to be involved in staphylococcal IE pathogenesis were tested. In parallel, the genotypic profiles of all isolates, obtained by microarray, were analyzed. No significant difference was observed between IE and bacteremia strains, regarding either phenotypic or genotypic univariate analyses, suggesting a multifactorial process. However, the discriminant analysis of principal components (DAPC), applied on microarray data, segregated IE and bacteremia isolates. The performance of this model was confirmed with an independent collection of IE and bacteremia isolates. Finally, a simple linear discriminant function based on a subset of 8 genetic markers retained valuable performance both in study collection and in the independent validation collection. At last, IE and bacteremia isolates were compared based on whole genome sequence data from a subset of 40 isolates. When applied to this dataset, DAPC confirmed a possible segregation between the two groups of isolates. All in all, this PhD work provides the proof of concept that bacterial characteristics may contribute to the occurrence of IE in patients with S. aureus bacteremia

La déficience intellectuelle (DI) est définie par un QI < 70. La DI, répartie en formes non syndromiques et en formes syndromiques, est observée dans 3 % de la population. Des anomalies chromosomiques sont identifiées dans 15 % des DI syndromiques. Les translocations chromosomiques réciproques (TR) apparemment équilibrées sont observées chez 1 individu sur 1000 et seul 6 % des patients avec une TR de novo apparemment équilibrée ont une DI. Plusieurs mécanismes chromosomiques peuvent expliquer la DI syndromique associée à une TR : (i) un microremaniement déséquilibré identifié par l'utilisation de techniques plus résolutives, (ii) la formation d'un gène de fusion, (iii) un effetde position, (iv) la modification d’une région soumise à une empreinte parentale, (v) une interruption d'un gène au niveau d'un ou des deux points de cassure, (vi) une mutation génique sans rapport avec la TR, (vii) ou encore une cause acquise ou multifactorielle. Nous rapportons l'étude de 12 patients avec DI et porteurs d'une TR de novo apparemment équilibrée. L'analyse systématique par puces à ADN de ces individus a été réalisée avec une résolution de 25 kb. Un déséquilibre infracytogénétique au niveau des points de cassure ou ailleurs dans le génome a été observé chez 3/12 patients. Chez les 9 patients sans anomalies sur puces à ADN, nous avons étudié les points de cassure des remaniements de novo apparemment équilibrés. En dehors de la technique de marche sur le chromosome par FISH, deux autres approches ont été mises en oeuvre : (i) l'Array-Painting qui correspond à l'hybridation sur puces à ADN de chacun des dérivés chromosomiques préalablement séparés par Cytométrie en Flux, (ii) et le séquençage haut débit (WGS - Whole Genome Sequencing). Grâce à l'Array-Painting, nous avons identifié (i) chez 2 patients, des interruptions de gènes pouvant expliquer leur phénotype, à savoir les gènes : KIF1A, AUTS2 et EPHA6 ; (ii) et chez 1 patiente, un point de cassure entraînant une dérégulation de la transcription du gène MEF2C. L'étude par WGS a permis (i) chez 1 patiente, de diagnostiquer un déséquilibre plus complexe que celui observé par puce à ADN ; (ii) chez 2 patients, de mettre en évidence unchromothripsis, qui pourrait avoir un impact dans les pathologies constitutionnelles par interruption de gènes et/ou par effet de position ; (iii) et chez 2 autres patients, de caractériser précisément les points de cassure. Ainsi, grâce aux résultats obtenus par ces différentes techniques, plusieurs mécanismes physiopathologiques responsables de DI sont mis en évidence permettant un conseil génétique adéquat. Cependant, aucun mécanisme chromosomique commun ne peut être identifié hormis le chromothripsis observé chez patients. Finalement, ce travail nous permet principalement de comparer les techniques mises en oeuvre qui se sont avérées complémentaires. En conclusion, nous proposons une démarche diagnostique pour explorer un remaniement chromosomique apparemment équilibré chez des patients à phénotype anormal / Intellectual disability (ID) is defined by an IQ <70. ID, observed in 3% of the population, and displays heterogeneous origins, including acquired etiology (toxicologic, pathologic, traumatic) or genetic disorders with non-syndromic and syndromic forms. Numerical or structural chromosomal abnormalities are observed in 15% of patients with ID. Reciprocal balanced chromosomal translocations (RT) are observed in one individual in 1000. However, only 6% of patients carrying a de novo apparently balanced RT present ID. The relation between these balanced rearrangements and ID could be explained by different mechanisms namely (i) subtle rearrangement, (ii) gene fusion, (iii) position effect, (iv) disturbance of parental imprinting, (v) gene disruption at the breakpoints, (vi) mutation in gene unrelated to the translocation, (vii) or acquired or multifactorial cause. We report a chromosomal study of 12 patients with DI and carrying a de novo apparently balanced reciprocal translocation. A systematic analysis by microarrays was performed in all individuals (using a resolution of 25 kb). For three patients, a microdeletion was observed at the breakpoints or elsewhere in the genome. For the 9 remaining cases, we hypothesize that the phenotype is due to a disruption of gene(s) located at the breakpoint(s). In this context, we studied the breakpoints of the apparently balanced de novo rearrangements in these patients. Outside FISH walking, two approaches have been implemented namely Array-Painting, which combines flow chromosome sorting in an attempt to isolate derivative chromosomes from each other and DNA microarrays as well as Whole Genome Sequencing (WGS). Using Array-Painting, we identified (i) in 2 patients, a gene disruptions: in the KIF1A, AUTS2 and EphA6 genes; (ii) and in 1 patient, a breakpoint resulting in deregulation of transcription of the gene MEF2C. The WGS technology has permitted (i) in 1 patient, to diagnose more complex imbalance than that observed by micro-array; (ii) in 2 patients, to show a chromothripsis, (iii) and 2 other patients, to characterize precisely breakpoints. In conclusion, taking together, these results highlight different physiopathological mechanisms responsible for DI allowing adequate genetic counseling. However, no common chromosomal mechanism can be identified except for chromothripsis observed in 2 patients. In addition, this work allows us especially to compare the used techniques which seem to be complementary. Finally, we propose a pipeline to elucidate the etiology of the abnormal phenotype in patients carrying an apparently balanced rearrangement

I denna studie undersöks värdet av diagnostik vid sällsynta sjukdomar hos unga individer. Då området är mångfacetterat studeras två fall med olika karaktär. Det första fallet undersöker värdet av screening för den sällsynta sjukdomen fenylketonuri (PKU) bland nyfödda, denna screening har utförts sedan 1960-talet. Det andra fallet fokuserar på en mer modern teknologisk utveckling och utvärderar värdet av införandet av helgenomsekvensering (WGS) som genetiskt test vid sökandet efter sällsynta sjukdomar.  Båda fallen använder sig av kostnadseffektivitetsanalys som metod där kostnader respektive hälsoeffekter estimeras för de utvärderade insatserna. Fallen skiljer sig åt med avseende på tillgängliga dataunderlag vilket innebär att tillvägagångssättet för att skatta kostnaderna och hälsoeffekterna är olika i de båda fallen. I fallet med PKU-screening används Markovmodellering där data från olika källor syntetiseras i en simuleringsmodell. I fallet med WGS-testning används i större utsträckning ett insamlat empiriskt datamaterial som utgörs av faktiskt uppmätta sjukvårdskostnader.  Resultaten i båda fallen indikerar att de diagnostiska metoderna har en rimlig kostnad i förhållande till hälsoeffekterna. Fall ett åskådliggör att dagens screening för PKU genererar ökade hälsoeffekter till lägre kostnader i jämförelse med att inte screena för PKU. För en kohort på 100 000 nyfödda barn blir den sammanlagda hälsoeffekten en ökning med 73 QALYs och screeningen medför samtidigt en besparing på 53 376 602 kr, sett över ett livstidsperspektiv. Fall två visar att WGS som första genetiskt test i genomsnitt minskar sjukvårdskostnaderna med 15 903 kr per individ jämfört med nuvarande vård och ökar samtidigt chansen till diagnos med 9,5 procentenheter (45,7%). Resultaten bör tolkas med viss försiktighet då de är förknippade med osäkerheter, men kan samtidigt användas som en del av det underlag beslutsfattare behöver för att fatta beslut om hur hälso- och sjukvårdens resurser ska prioriteras. / This study examines the value of diagnostics in rare diseases in young individuals. As the field is varied, two cases with different character are studied. The first case examines the value of screening for the rare disease phenylketonuria (PKU) among newborns, this screening has been performed since the 1960s. The second case focuses on a more modern technological development and evaluates the value of the introduction of whole genome sequencing (WGS) as a genetic test in the search for rare diseases.  Both cases utilize the method of cost-effectiveness analysis where costs and health effects are estimated for the evaluated measures. The cases differ regarding available data, which means that the approach to estimating costs and health effects is different in the two cases. In the case of PKU- screening, Markov modeling is used where data from different sources are synthesized in a simulation model. In the case of WGS-testing, an empirical data material is used to a greater extent, which is based on actually measured healthcare costs.  The results in both cases indicate that the diagnostic methods have a reasonable cost in relation to the health effects. Case one illustrates that today's screening for PKU generates increased health effects at lower costs compared to not screening for PKU. For a cohort of 100 000 newborns, the total health effect will be an increase of 73 QALYs and the screening will also result in cost- savings of SEK 53 376 602, seen from a lifetime perspective. Case two shows that WGS used as an initial genetic test on average reduces healthcare costs by SEK 15 903 per individual compared with current care and at the same time increases the chance of diagnosis by 9.5 percentage points (45.7%). The results should be interpreted with some caution as they are associated with some uncertainties, but can still be used as part of the basis on which decision-makers need to make decisions on how health care resources should be prioritized.

<p>Heterosis, or hybrid vigor, is a phenomenon observed in both plant and animal systems where hybrid offspring perform better when compared to their parents. For hybrid plants, this can result in increased biomass, crop yields, and vigor when compared to the inbred parents. Even though heterosis has been used in agriculture for over a century, the molecular mechanisms that result in hybrid vigor remain elusive even after years of investigation. A molecular understanding of heterosis is desirable because it will speed up the process of breeding compatible inbred lines for developing hybrid seeds, and it will provide us with the knowledge to potentially engineer inbred lines that can mimic the beneficial phenotypic effects of heterosis, eliminating the need for farmers to buy new hybrid seeds every year. The goal of this research project is to identify genes that are required for heterotic phenotypes in maize. Our working hypothesis is that a mutation in genes that are essential for heterosis will cause an altered heterotic phenotype in hybrid maize plants. To test this hypothesis, we applied combined approaches of EMS mutagenesis, trait phenotyping in field and controlled conditions, bulk segregant analysis, whole genome sequencing, and bioinformatics analysis. First, we applied a forward genetics approach to identify mutant hybrids with altered heterosis and detected potential causal genes <em>via</em> whole genome sequencing. We identified one mutation occurring in a protein coding gene (gene ID <em>Zm00001eb305590</em>) located in a region of interest on chromosome 7, whose genotypes across various samples assayed fit the observed segregation pattern of hybrid traits. This mutation leads to a moderate or high-level codon change, indicating that this gene may play a role in mediating heterosis in maize. By investigating this gene with further studies, the learned knowledge could speed up the process of hybrid maize breeding by selecting compatible inbred lines through sequencing or by engineering hybrids that have favorable alleles for this gene.</p>

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