HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

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.

O gênero Xylella é composto de uma única espécie, Xylella fastidiosa, bactéria Gram-negativa, não flagelada, que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo. Em algumas dessas plantas, a bactéria é considerada agente causal de doenças, como a Clorose Variegada do Citros em laranjeiras, a Doença de Pierce das videiras e escaldadura da folha de cafeeiro. Onze diferentes cepas de X. fastidiosa, isoladas de distintos hospedeiros, já tiveram seus genomas sequenciados, entre essas, as cepas 9a5c, isolada de laranjeira, e Temecula 1, isolada de videira. Análises desses genomas indicam uma razoável variabilidade entre suas respectivas sequências e evidenciam vários genes associados a mecanismos de virulência e patogenicidade desta bactéria. No presente trabalho descrevemos o sequenciamento, a montagem e a anotação dos genomas das cepas U24d e Fb7, isoladas de laranjeiras, e da cepa 3124 isolada de cafeeiro, os quais apresentam, respectivamente 2.681.334 pb, 2.733.974 pb e 2.748.594 pb. Destas, apenas a cepa U24d apresenta um plasmídeo, o qual é idêntico ao pXF51 previamente identificado na cepa 9a5c. O genoma da cepa U24d é praticamente colinear ao genoma da cepa 9a5c enquanto que os genomas das cepas Fb7 e 3124 apresentaram maior colinearidade com a cepa Temecula1. Entre as diversas alterações encontradas nas análises comparativas destes genomas, destacamos a inserção no gene pilQ verificada no genoma da cepa U24d. Essa mutação causa ausência do pilus do tipo IV com consequente deficiência na motilidade twitching, sendo que plantas infectadas com a cepa U24d apresentam sintomas localizados restritos ao ponto de inoculação. Na cepa Fb7, detectamos a ausência de formação de biofilme no cultivo in vitro possivelmente devido ausência da expressão dos transcritos de mrkD e pspA, que codificam respectivamente adesina do pilus curto e adesina similar à hemaglutinina. Postulamos que estes genes não são expressos em decorrência de um defeito na via de sinalização de DSF (Fator de Sinalização Difusível) reflexo de uma mutação em rpfC no genoma de Fb7. Assim como as demais cepas de X. fastidiosa, também os genomas de U24d, Fb7 e 3124 apresentaram elevado conteúdo de Elementos Genéticos Móveis (EGM), que aparecem em maior número nas cepas sul-americanas. Os estudos do pangenoma de X. fastidiosa mostraram que essa espécie tem um genoma aberto e grande parte dos genes de EGMs correspondem a genes acessórios. A grande quantidade de EGMs em X. fastidiosa pode estar relacionada a falta do sistema CRISPR/cas completo, um provável resultado de eventos de erosão do genoma desta espécie. A inferência filogenética por MSLA mostrou uma clara distinção dos grupos de cepas da América do Norte em relação às do Sul, sugerindo a ocorrência de mais eventos de recombinações genéticas nas cepas sul-americanas, provavelmente pela falta de isolamento geográfico. Assim, é possível que as cepas norte e sul-americanas sofreram divergência alopátrica e simpátrica, respectivamente. / The genus Xylella consists of a single species, Xylella fastidiosa, a Gram-negative and non-flagellated bacterium that colonizes the xylem of a diversity of cultivated and wild plants in several parts of the world. In some of these plants, this bacterium is considered causal agent of diseases such as the Citrus Variegated Cholorosis in orange trees, Pierce\'s Disease of grapevines and coffee leaf scald. Eleven different strains of X. fastidiosa isolated from different hosts had their genomes sequenced, including 9a5c and Temecula1 strains, respectively isolated from orange tree and grapevine. Analyses of these genomes indicate a reasonable variability in their sequences and showed several genes associated with pathogenicity and virulence mechanisms of this bacterium. In this work we describe the genome sequencing, assembly and annotation of the strains U24d and Fb7, isolated from orange trees, and 3124 isolated from coffee, which have, respectively, 2,681,334 bp, 2,733,974 bp and 2,748,594 bp. Of these, only strain U24d has a plasmid, identical to pXF51 from strain 9a5c. The genome of U24d strain is almost collinear to the genome of strain 9a5c while the genomes of strains Fb7 and 3124 had higher collinearity to Temecula1 strain. Among many changes found in the comparative analysis of these genomes, we highlight an on insertion in pilQ gene that was found in U24d strain genome. This mutation causes lack of type IV pilus with a consequent deficiency in twitching motility. Moreover orange trees infected with U24d strain showed localized symptoms near to the inoculation point. We verified that Fb7 strain does not form biofilm in vitro possibly due to the absence of expression of mrkD and pspA transcripts, which encode, respectively, a short pilus adhesin and a hemagglutinin-like adhesin. We postulate that these genes are not expressed due to a defect in the signaling pathway of DSF (Diffusible Signal Factor) reflecting a mutation on rpfC in the Fb7 genome. Similarly to other X. fastidiosa strains, the genomes of U24d, Fb7 and 3124 also showed high content of mobile genetic elements (MGE), which appear in larger numbers in South American strains. Pan genome studies of X. fastidiosa showed that this species has a open genome and that most of MGE genes correspond to accessory genes. The large number of MGE in X. fastidiosa may be related to the lack of a complete system CRISPR/cas, likely a result of erosion events of the genome of this species. The phylogenetic reconstruction by MLSA showed a clear distinction between groups of strains from North and South America, suggesting the occurrence of more recombination events in South American strains, probably due to lack of geographical isolation. Thus it is possible that North and South American strains underwent allopatric and sympatric divergence, respectively.

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

No description available.

This work documents the first case of idiopathic AA amyloidosis in humans caused by mutation in the promoter region of SAA1 gene. Knowledge of the mechanism of the disease may be an indication for targeted treatment in the future. Mutations in the SAA1 promoter should be considered in all cases of idiopathic forms of AA amyloidosis in which neither the immune nor the inflammatory component of the disease are clearly present.

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.