Genome-wide association for abdominal subcutaneous and visceral adipose reveals a novel locus for visceral fat in women.

Body fat distribution, particularly centralized obesity, is associated with metabolic risk above and beyond total adiposity. We performed genome-wide association of abdominal adipose depots quantified using computed tomography (CT) to uncover novel loci for body fat distribution among participants of European ancestry. Subcutaneous and visceral fat were quantified in 5,560 women and 4,997 men from 4 population-based studies. Genome-wide genotyping was performed using standard arrays and imputed to ~2.5 million Hapmap SNPs. Each study performed a genome-wide association analysis of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), VAT adjusted for body mass index, and VAT/SAT ratio (a metric of the propensity to store fat viscerally as compared to subcutaneously) in the overall sample and in women and men separately. A weighted z-score meta-analysis was conducted. For the VAT/SAT ratio, our most significant p-value was rs11118316 at LYPLAL1 gene (p = 3.1 × 10E-09), previously identified in association with waist-hip ratio. For SAT, the most significant SNP was in the FTO gene (p = 5.9 × 10E-08). Given the known gender differences in body fat distribution, we performed sex-specific analyses. Our most significant finding was for VAT in women, rs1659258 near THNSL2 (p = 1.6 × 10-08), but not men (p = 0.75). Validation of this SNP in the GIANT consortium data demonstrated a similar sex-specific pattern, with observed significance in women (p = 0.006) but not men (p = 0.24) for BMI and waist circumference (p = 0.04 [women], p = 0.49 [men]). Finally, we interrogated our data for the 14 recently published loci for body fat distribution (measured by waist-hip ratio adjusted for BMI); associations were observed at 7 of these loci. In contrast, we observed associations at only 7/32 loci previously identified in association with BMI; the majority of overlap was observed with SAT. Genome-wide association for visceral and subcutaneous fat revealed a SNP for VAT in women. More refined phenotypes for body composition and fat distribution can detect new loci not previously uncovered in large-scale GWAS of anthropometric traits.

Characterization of a genomic signature of pregnancy identified in the breast.

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer.

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews.

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.

Mechanisms of functional T-cell deficiency in melanoma patients

SUMMARYAs a result of evolution, humans are equipped with an intricate but very effective immune system with multiple defense mechanisms primarily providing protection from infections. This system comprises various cell types, including T-lymphocytes, which are able to recognize and directly kill infected cells. T-cells are not only able to recognize cells carrying foreign antigens, such as virus-infected cells, but also autologous cells. In autoimmune diseases, e.g. multiple sclerosis, T- cells attack autologous cells and cause the destruction of healthy tissue. To prevent aberrant immune reactions, but also to prevent damage caused by an overreacting immune response against foreign targets, there are multiple systems in place that attenuate T-cell responses.By contrast, anti-self immune responses may be highly welcome in malignant diseases. It has been demonstrated that activated T-cells are able to recognize and lyse tumor cells, and may even lead to successful cure of cancer patients. Through vaccination, and especially with the help of powerful adjuvants, frequencies of tumor-reactive T-cells can be augmented drastically. However, the efficacy of anti-tumor responses is diminished by the same checks and balances preventing the human body from harm induced by overly activated T-cells in infections.In the context of my thesis, we studied spontaneous and vaccination induced T-cell responses in melanoma patients. The aim of my studies was to identify situations of T-cell suppression, and pinpoint immune suppressive mechanisms triggered by malignant diseases. We applied recently developed techniques such as multiparameter flow cytometry and gene arrays, allowing the characterization of tumor-reactive T-cells directly ex vivo. In our project, we determined functional capabilities, protein expression, and gene expression profiles of small numbers of T- cells from metastatic tissue and blood obtained from healthy donors and melanoma patients. We found evidence that tumor-specific T-cells were functionally efficient effector cells in peripheral blood, but severely exhausted in metastatic tissue. Our molecular screening revealed the upregulation of multiple inhibitory receptors on tumor-specific T-cells, likely implied in T-cell exhaustion. Functional attenuation of tumor-specific T-cells via inhibitory receptors depended on the anatomical location and immune suppressive mechanisms in the tumor microenvironment, which appeared more important than self-tolerance and anergy mechanisms. Our data reveal novel potential targets for cancer therapy, and contribute to the understanding of cancer biology.RÉSUMÉAu cours de l'évolution, les êtres humains se sont vus doter d'un système immunitaire complexe mais très efficace, avec de multiples mécanismes de défense, principalement contre les infections. Ce système comprend différents types de cellules, dont les lymphocytes Τ qui sont capables de reconnaître et de tuer directement des cellules infectées. Les cellules Τ reconnaissent non seulement des cellules infectées par des virus, mais également des cellules autologues. Dans le cas de maladies auto-immunes, comme par exemple la sclérose en plaques, les cellules Τ s'attaquent à des cellules autologues, ce qui engendre la destruction des tissus sains. Il existe plusieurs systèmes de contrôle des réponses Τ afin de minimiser les réactions immunitaires aberrantes et d'empêcher les dégâts causés par une réponse immunitaire trop importante contre une cible étrangère.Dans le cas de maladies malignes en revanche, une réponse auto-immune peut être avantageuse. Il a été démontré que les lymphocytes Τ étaient également capables de reconnaître et de tuer des cellules tumorales, pouvant même mener à la guérison d'un patient cancéreux. La vaccination peut augmenter fortement la fréquence des cellules Τ réagissant contre une tumeur, particulièrement si elle est combinée avec des adjuvants puissants. Cependant, l'efficacité d'une réponse antitumorale est atténuée par ces mêmes mécanismes de contrôle qui protègent le corps humain des dégâts causés par des cellules Τ activées trop fortement pendant une infection.Dans le cadre de ma recherche de thèse, nous avons étudié les réponses Τ spontanées et induites par la vaccination dans des patients atteints du mélanome. Le but était d'identifier des conditions dans lesquelles les réponses des cellules Τ seraient atténuées, voire inhibées, et d'élucider les mécanismes de suppression immunitaire engendrés par le cancer. Par le biais de techniques nouvelles comprenant la cryométrie de flux et l'analyse globale de l'expression génique à partir d'un nombre minimal de cellules, il nous fut possible de caractériser des cellules Τ réactives contre des tumeurs directement ex vivo. Nous avons examiné les profiles d'expression de gènes et de protéines, ainsi que les capacités fonctionnelles des cellules Τ isolées à partir de tissus métastatiques et à partir du sang de patients. Nos résultats indiquent que les cellules Τ spécifiques aux antigènes tumoraux sont fonctionnelles dans le sang, mais qu'elles sont épuisées dans les tissus métastatiques. Nous avons découvert dans les cellules Τ antitumorales une augmentation de l'expression des récepteurs inhibiteurs probablement impliqués dans l'épuisement de ces lymphocytes T. Cette expression particulière de récepteurs inhibiteurs dépendrait donc de leur localisation anatomique et des mécanismes de suppression existant dans l'environnement immédiat de la tumeur. Nos données révèlent ainsi de nouvelles cibles potentielles pour l'immunothérapie du cancer et contribuent à la compréhension biologique du cancer.

Measuring brain perfusion with intravoxel incoherent motion (IVIM): Initial clinical experience.

PURPOSE: To evaluate the feasibility of intravoxel incoherent motion (IVIM) perfusion measurements in the brain with currently available imaging systems. MATERIALS AND METHODS: We acquired high in-plane resolution (1.2 × 1.2 mm(2) ) diffusion-weighted images with 16 different values of b ranging from 0 to 900 s/mm(2) , in three orthogonal directions, on 3T systems with a 32-multichannel receiver head coil. IVIM perfusion maps were extracted by fitting a double exponential model of signal amplitude decay. Regions of interest were drawn in pathological and control regions, where IVIM perfusion parameters were compared to the corresponding dynamic susceptibility contrast (DSC) parameters. RESULTS: Hyperperfusion was found in the nonnecrotic or cystic part of two histologically proven glioblastoma multiforme and in two histologically proven glioma WHO grade III, as well as in a brain metastasis of lung adenocarcinoma, in a large meningioma, and in a case of ictal hyperperfusion. A monoexponential decay was found in a territory of acute ischemia, as well as in the necrotic part of a glioblastoma. The IVIM perfusion fraction f correlated well with DSC CBV. CONCLUSION: Our initial report suggests that high-resolution brain perfusion imaging is feasible with IVIM in the current clinical setting. J. Magn. Reson. Imaging 2014;39:624-632. © 2013 Wiley Periodicals, Inc.

Inexperienced sonographers can successfully visualize and assess a three-dimensional image of the fetal face using a standardized ultrasound protocol.

Introduction: A standardized three-dimensional ultrasonographic (3DUS) protocol is described that allows fetal face reconstruction. Ability to identify cleft lip with 3DUS using this protocol was assessed by operators with minimal 3DUS experience. Material and Methods: 260 stored volumes of fetal face were analyzed using a standardized protocol by operators with different levels of competence in 3DUS. The outcomes studied were: (1) the performance of post-processing 3D face volumes for the detection of facial clefts; (2) the ability of a resident with minimal 3DUS experience to reconstruct the acquired facial volumes, and (3) the time needed to reconstruct each plane to allow proper diagnosis of a cleft. Results: The three orthogonal planes of the fetal face (axial, sagittal and coronal) were adequately reconstructed with similar performance when acquired by a maternal-fetal medicine specialist or by residents with minimal experience (72 vs. 76%, p = 0.629). The learning curve for manipulation of 3DUS volumes of the fetal face corresponds to 30 cases and is independent of the operator's level of experience. Discussion: The learning curve for the standardized protocol we describe is short, even for inexperienced sonographers. This technique might decrease the length of anatomy ultrasounds and improve the ability to visualize fetal face anomalies.

MGMT methylation status: the advent of stratified therapy in glioblastoma?

Glioblastomas are the most malignant gliomas with median survival times of only 15 months despite modern therapies. All standard treatments are palliative. Pathogenetic factors are diverse, hence, stratified treatment plans are warranted considering the molecular heterogeneity among these tumors. However, most patients are treated with "one fits all" standard therapies, many of them with minor response and major toxicities. The integration of clinical and molecular information, now becoming available using new tools such as gene arrays, proteomics, and molecular imaging, will take us to an era where more targeted and effective treatments may be implemented. A first step towards the design of such therapies is the identification of relevant molecular mechanisms driving the aggressive biological behavior of glioblastoma. The accumulation of diverse aberrations in regulatory processes enables tumor cells to bypass the effects of most classical therapies available. Molecular alterations underlying such mechanisms comprise aberrations on the genetic level, such as point mutations of distinct genes, or amplifications and deletions, while others result from epigenetic modifications such as aberrant methylation of CpG islands in the regulatory sequence of genes. Epigenetic silencing of the MGMT gene encoding a DNA repair enzyme was recently found to be of predictive value in a randomized clinical trial for newly diagnosed glioblastoma testing the addition of the alkylating agent temozolomide to standard radiotherapy. Determination of the methylation status of the MGMT promoter may become the first molecular diagnostic tool to identify patients most likely to respond that will allow individually tailored therapy in glioblastoma. To date, the test for the MGMT-methylation status is the only tool available that may direct the choice for alkylating agents in glioblastoma patients, but many others may hopefully become part of an arsenal to stratify patients to respective targeted therapies within the next years.

La fonction de la carte géographique : approche épistémologique ternaire

Under the referential of a ternary logic, with the two orthogonal concepts of information/signification, mediated by the third-included, the diagonal organizer of the cartographic communication, this article aims to cladify the pragmatical aspects of the map functionnality as a communication object. The interaction beween the meaning of its information and the information reciprocally contained in its signification, within a specified context and pertinence, implies a strong level of redundancy shared by producers and users of the map, a same system of scientific and ideological values to pass on the cartographic message. We are far from the objective cartographer who would fade away so to let the facts speaks "objectively" by themselves.

Protective role of early aquaporin 4 induction against postischemic edema formation.

Aquaporin 4 (AQP4) is a water channel involved in water movements across the cell membrane and is spatially organized on the cell surface in orthogonal array particles (OAPs). Its role in edema formation or resolution after stroke onset has been studied mainly at late time points. We have shown recently that its expression is rapidly induced after ischemia coinciding in time with an early swelling of the ischemic hemisphere. There are two isoforms of AQP4: AQP4-M1 and AQP4-M23. The ratio of these isoforms influences the size of the OAPs but the functional impact is not known. The role of the early induction of AQP4 is not yet known. Thrombin preconditioning in mice provides a useful model to study endogenous protective mechanisms. Using this model, we provide evidence for the first time that the early induction of AQP4 may contribute to limit the formation of edema and that the AQP4-M1 isoform is predominantly induced in the ischemic tissue at this time point. Although it prevents edema formation, the early induction of the AQP4 expression does not prevent the blood-brain barrier disruption, suggesting an effect limited to the prevention of edema formation possibly by removing of water from the tissue.

A microRNA profile of human CD8.

BackgroundRecently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8+CD25+ Treg cells and the impact of microRNAs on molecules associated with immune regulation.MethodsWe purified human natural CD8+ Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA `signature¿ for CD8+CD25+FOXP3+CTLA-4+ natural Treg cells. We used the `TargetScan¿ and `miRBase¿ bioinformatics programs to identify potential target sites for these microRNAs in the 3¿-UTR of important Treg cell-associated genes.ResultsThe human CD8+CD25+ natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.ConclusionsWe are examining the biological relevance of this `signature¿ by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Qu'attendre du futur du diagnostic en allergologie [What to expect from allergy tests?].

Diagnosis in allergology is facing novel challenges because of the availability not only of purified or recombinant allergens, but also of multitests such as allergen micro-arrays. These new diagnostic opportunities contribute to a better understanding of crossreactivities between respiratory and food allergens. In comparison to current diagnosis based on whole allergen extracts, this novel generation of specific IgE tests is expected to provide better information on the risk of reaction to allergens as well as on its severity. However these new technologies are expensive, and will have to be carefully analyzed in terms of medical usefulness and public health costs.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

Computational and statistical analysis of copy number variants in normal and cancer genomes

AbstractAlthough the genomes from any two human individuals are more than 99.99% identical at the sequence level, some structural variation can be observed. Differences between genomes include single nucleotide polymorphism (SNP), inversion and copy number changes (gain or loss of DNA). The latter can range from submicroscopic events (CNVs, at least 1kb in size) to complete chromosomal aneuploidies. Small copy number variations have often no (lethal) consequences to the cell, but a few were associated to disease susceptibility and phenotypic variations. Larger re-arrangements (i.e. complete chromosome gain) are frequently associated with more severe consequences on health such as genomic disorders and cancer. High-throughput technologies like DNA microarrays enable the detection of CNVs in a genome-wide fashion. Since the initial catalogue of CNVs in the human genome in 2006, there has been tremendous interest in CNVs both in the context of population and medical genetics. Understanding CNV patterns within and between human populations is essential to elucidate their possible contribution to disease. But genome analysis is a challenging task; the technology evolves rapidly creating needs for novel, efficient and robust analytical tools which need to be compared with existing ones. Also, while the link between CNV and disease has been established, the relative CNV contribution is not fully understood and the predisposition to disease from CNVs of the general population has not been yet investigated.During my PhD thesis, I worked on several aspects related to CNVs. As l will report in chapter 3, ! was interested in computational methods to detect CNVs from the general population. I had access to the CoLaus dataset, a population-based study with more than 6,000 participants from the Lausanne area. All these individuals were analysed on SNP arrays and extensive clinical information were available. My work explored existing CNV detection methods and I developed a variety of metrics to compare their performance. Since these methods were not producing entirely satisfactory results, I implemented my own method which outperformed two existing methods. I also devised strategies to combine CNVs from different individuals into CNV regions.I was also interested in the clinical impact of CNVs in common disease (chapter 4). Through an international collaboration led by the Centre Hospitalier Universitaire Vaudois (CHUV) and the Imperial College London I was involved as a main data analyst in the investigation of a rare deletion at chromosome 16p11 detected in obese patients. Specifically, we compared 8,456 obese patients and 11,856 individuals from the general population and we found that the deletion was accounting for 0.7% of the morbid obesity cases and was absent in healthy non- obese controls. This highlights the importance of rare variants with strong impact and provides new insights in the design of clinical studies to identify the missing heritability in common disease.Furthermore, I was interested in the detection of somatic copy number alterations (SCNA) and their consequences in cancer (chapter 5). This project was a collaboration initiated by the Ludwig Institute for Cancer Research and involved other groups from the Swiss Institute of Bioinformatics, the CHUV and Universities of Lausanne and Geneva. The focus of my work was to identify genes with altered expression levels within somatic copy number alterations (SCNA) in seven metastatic melanoma ceil lines, using CGH and SNP arrays, RNA-seq, and karyotyping. Very few SCNA genes were shared by even two melanoma samples making it difficult to draw any conclusions at the individual gene level. To overcome this limitation, I used a network-guided analysis to determine whether any pathways, defined by amplified or deleted genes, were common among the samples. Six of the melanoma samples were potentially altered in four pathways and five samples harboured copy-number and expression changes in components of six pathways. In total, this approach identified 28 pathways. Validation with two external, large melanoma datasets confirmed all but three of the detected pathways and demonstrated the utility of network-guided approaches for both large and small datasets analysis.RésuméBien que le génome de deux individus soit similaire à plus de 99.99%, des différences de structure peuvent être observées. Ces différences incluent les polymorphismes simples de nucléotides, les inversions et les changements en nombre de copies (gain ou perte d'ADN). Ces derniers varient de petits événements dits sous-microscopiques (moins de 1kb en taille), appelés CNVs (copy number variants) jusqu'à des événements plus large pouvant affecter des chromosomes entiers. Les petites variations sont généralement sans conséquence pour la cellule, toutefois certaines ont été impliquées dans la prédisposition à certaines maladies, et à des variations phénotypiques dans la population générale. Les réarrangements plus grands (par exemple, une copie additionnelle d'un chromosome appelée communément trisomie) ont des répercutions plus grave pour la santé, comme par exemple dans certains syndromes génomiques et dans le cancer. Les technologies à haut-débit telle les puces à ADN permettent la détection de CNVs à l'échelle du génome humain. La cartographie en 2006 des CNV du génome humain, a suscité un fort intérêt en génétique des populations et en génétique médicale. La détection de différences au sein et entre plusieurs populations est un élément clef pour élucider la contribution possible des CNVs dans les maladies. Toutefois l'analyse du génome reste une tâche difficile, la technologie évolue très rapidement créant de nouveaux besoins pour le développement d'outils, l'amélioration des précédents, et la comparaison des différentes méthodes. De plus, si le lien entre CNV et maladie a été établit, leur contribution précise n'est pas encore comprise. De même que les études sur la prédisposition aux maladies par des CNVs détectés dans la population générale n'ont pas encore été réalisées.Pendant mon doctorat, je me suis concentré sur trois axes principaux ayant attrait aux CNV. Dans le chapitre 3, je détaille mes travaux sur les méthodes d'analyses des puces à ADN. J'ai eu accès aux données du projet CoLaus, une étude de la population de Lausanne. Dans cette étude, le génome de plus de 6000 individus a été analysé avec des puces SNP et de nombreuses informations cliniques ont été récoltées. Pendant mes travaux, j'ai utilisé et comparé plusieurs méthodes de détection des CNVs. Les résultats n'étant pas complètement satisfaisant, j'ai implémenté ma propre méthode qui donne de meilleures performances que deux des trois autres méthodes utilisées. Je me suis aussi intéressé aux stratégies pour combiner les CNVs de différents individus en régions.Je me suis aussi intéressé à l'impact clinique des CNVs dans le cas des maladies génétiques communes (chapitre 4). Ce projet fut possible grâce à une étroite collaboration avec le Centre Hospitalier Universitaire Vaudois (CHUV) et l'Impérial College à Londres. Dans ce projet, j'ai été l'un des analystes principaux et j'ai travaillé sur l'impact clinique d'une délétion rare du chromosome 16p11 présente chez des patients atteints d'obésité. Dans cette collaboration multidisciplinaire, nous avons comparés 8'456 patients atteint d'obésité et 11 '856 individus de la population générale. Nous avons trouvés que la délétion était impliquée dans 0.7% des cas d'obésité morbide et était absente chez les contrôles sains (non-atteint d'obésité). Notre étude illustre l'importance des CNVs rares qui peuvent avoir un impact clinique très important. De plus, ceci permet d'envisager une alternative aux études d'associations pour améliorer notre compréhension de l'étiologie des maladies génétiques communes.Egalement, j'ai travaillé sur la détection d'altérations somatiques en nombres de copies (SCNA) et de leurs conséquences pour le cancer (chapitre 5). Ce projet fut une collaboration initiée par l'Institut Ludwig de Recherche contre le Cancer et impliquant l'Institut Suisse de Bioinformatique, le CHUV et les Universités de Lausanne et Genève. Je me suis concentré sur l'identification de gènes affectés par des SCNAs et avec une sur- ou sous-expression dans des lignées cellulaires dérivées de mélanomes métastatiques. Les données utilisées ont été générées par des puces ADN (CGH et SNP) et du séquençage à haut débit du transcriptome. Mes recherches ont montrées que peu de gènes sont récurrents entre les mélanomes, ce qui rend difficile l'interprétation des résultats. Pour contourner ces limitations, j'ai utilisé une analyse de réseaux pour définir si des réseaux de signalisations enrichis en gènes amplifiés ou perdus, étaient communs aux différents échantillons. En fait, parmi les 28 réseaux détectés, quatre réseaux sont potentiellement dérégulés chez six mélanomes, et six réseaux supplémentaires sont affectés chez cinq mélanomes. La validation de ces résultats avec deux larges jeux de données publiques, a confirmée tous ces réseaux sauf trois. Ceci démontre l'utilité de cette approche pour l'analyse de petits et de larges jeux de données.Résumé grand publicL'avènement de la biologie moléculaire, en particulier ces dix dernières années, a révolutionné la recherche en génétique médicale. Grâce à la disponibilité du génome humain de référence dès 2001, de nouvelles technologies telles que les puces à ADN sont apparues et ont permis d'étudier le génome dans son ensemble avec une résolution dite sous-microscopique jusque-là impossible par les techniques traditionnelles de cytogénétique. Un des exemples les plus importants est l'étude des variations structurales du génome, en particulier l'étude du nombre de copies des gènes. Il était établi dès 1959 avec l'identification de la trisomie 21 par le professeur Jérôme Lejeune que le gain d'un chromosome supplémentaire était à l'origine de syndrome génétique avec des répercussions graves pour la santé du patient. Ces observations ont également été réalisées en oncologie sur les cellules cancéreuses qui accumulent fréquemment des aberrations en nombre de copies (telles que la perte ou le gain d'un ou plusieurs chromosomes). Dès 2004, plusieurs groupes de recherches ont répertorié des changements en nombre de copies dans des individus provenant de la population générale (c'est-à-dire sans symptômes cliniques visibles). En 2006, le Dr. Richard Redon a établi la première carte de variation en nombre de copies dans la population générale. Ces découvertes ont démontrées que les variations dans le génome était fréquentes et que la plupart d'entre elles étaient bénignes, c'est-à-dire sans conséquence clinique pour la santé de l'individu. Ceci a suscité un très grand intérêt pour comprendre les variations naturelles entre individus mais aussi pour mieux appréhender la prédisposition génétique à certaines maladies.Lors de ma thèse, j'ai développé de nouveaux outils informatiques pour l'analyse de puces à ADN dans le but de cartographier ces variations à l'échelle génomique. J'ai utilisé ces outils pour établir les variations dans la population suisse et je me suis consacré par la suite à l'étude de facteurs pouvant expliquer la prédisposition aux maladies telles que l'obésité. Cette étude en collaboration avec le Centre Hospitalier Universitaire Vaudois a permis l'identification d'une délétion sur le chromosome 16 expliquant 0.7% des cas d'obésité morbide. Cette étude a plusieurs répercussions. Tout d'abord elle permet d'effectuer le diagnostique chez les enfants à naître afin de déterminer leur prédisposition à l'obésité. Ensuite ce locus implique une vingtaine de gènes. Ceci permet de formuler de nouvelles hypothèses de travail et d'orienter la recherche afin d'améliorer notre compréhension de la maladie et l'espoir de découvrir un nouveau traitement Enfin notre étude fournit une alternative aux études d'association génétique qui n'ont eu jusqu'à présent qu'un succès mitigé.Dans la dernière partie de ma thèse, je me suis intéressé à l'analyse des aberrations en nombre de copies dans le cancer. Mon choix s'est porté sur l'étude de mélanomes, impliqués dans le cancer de la peau. Le mélanome est une tumeur très agressive, elle est responsable de 80% des décès des cancers de la peau et est souvent résistante aux traitements utilisés en oncologie (chimiothérapie, radiothérapie). Dans le cadre d'une collaboration entre l'Institut Ludwig de Recherche contre le Cancer, l'Institut Suisse de Bioinformatique, le CHUV et les universités de Lausanne et Genève, nous avons séquencés l'exome (les gènes) et le transcriptome (l'expression des gènes) de sept mélanomes métastatiques, effectués des analyses du nombre de copies par des puces à ADN et des caryotypes. Mes travaux ont permis le développement de nouvelles méthodes d'analyses adaptées au cancer, d'établir la liste des réseaux de signalisation cellulaire affectés de façon récurrente chez le mélanome et d'identifier deux cibles thérapeutiques potentielles jusqu'alors ignorées dans les cancers de la peau.

Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.