Parcela considerável de pacientes com distúrbios de crescimento não têm a causa de seus quadros clínicos estabelecida, incluindo aproximadamente 50% dos pacientes com diagnóstico clínico de síndrome de Silver−Russell (SRS) e 10-20% dos pacientes com síndrome de Beckwith-Wiedemann (BWS). O objetivo deste estudo foi investigar as causas genéticas e epigenéticas de distúrbios de crescimento, de etiologia desconhecida, numa contribuição para o entendimento de mecanismos que regulam o crescimento. O estudo compreendeu: (1) a investigação de microdesequilíbrios cromossômicos, por aCGH; (2) a análise do perfil de expressão alelo-específica de genes sujeitos a imprinting (IG), por pirossequenciamento (PSQ) ou sequenciamento de Sanger; (3) a investigação do padrão de metilação global em pacientes com restrição de crescimento, utilizando microarray de metilação. A casuística constituiu-se de 41 pacientes não aparentados, com distúrbios de crescimento, de etiologia desconhecida: (1) 25, com hipótese diagnóstica de SRS; (2) seis, com restrição de crescimento intrauterino e peso ao nascimento abaixo do 10º percentil, associados a outros sinais clínicos; (3) sete, com hipótese diagnóstica de BWS; e (4) três, com macrossomia pré-natal ou pós-natal, associada a outros sinais. A investigação de microdesequilíbrios cromossômicos foi realizada em 40 pacientes. Foram detectadas 58 variantes raras em 30/40 pacientes (75%): 40 foram consideradas provavelmente benignas (18 pacientes, 45%), 12, com efeito patogênico desconhecido (11 pacientes, 27,5%), duas, provavelmente patogênicas (um paciente, 2,5%) e quatro, patogênicas (três pacientes, 7,5%). Essas frequências são comparáveis àquelas descritas em estudos que investigaram CNV em grupos de pacientes com distúrbios de crescimento e outras alterações congênitas, incluindo SRS, e mostram a importância da investigação de microdesequilíbrios cromossômicos nesses pacientes. A diversidade dos microdesequilíbrios cromossômicos identificados é reflexo da heterogeneidade clínica das casuísticas. Neste estudo, muitos dos pacientes com hipótese diagnóstica de SRS e BWS apresentavam sinais clínicos atípicos, explicando a ausência neles das alterações (epi)genéticas que causam essas síndromes. A identificação de CNV características de outras síndromes reflete a sobreposição de sinais clínicos com BWS e SRS. A análise do perfil de expressão alelo-específica de IG foi realizada em um subgrupo de 18 pacientes com restrição de crescimento. Trinta IG com função em proliferação celular, crescimento fetal ou neurodesenvolvimento foram inicialmente selecionados. Após seleção de SNP transcritos com alta frequência na população, genotipagem de pacientes, genitores e indivíduos controle, determinação da expressão dos IG em sangue periférico e seu padrão de expressão (mono ou bialélico), 13 IG, expressos no sangue, tiveram a expressão alelo-específica avaliada, sete deles por PSQ e seis por sequenciamento de Sanger. Alterações no perfil de expressão de dois genes, de expressão normalmente paterna, foram detectadas em 4/18 pacientes (22%). Este estudo é o primeiro a utilizar pirossequenciamento e sequenciamento de Sanger na avaliação do perfil de expressão alelo-específica de IG, em pacientes com restrição de crescimento. Apesar de terem limitações, ambas as técnicas mostraram-se robustas e revelaram alterações de expressão alélica interessantes; entretanto, a relação dessas alterações com o quadro clínico dos pacientes permanece por esclarecer. A investigação da metilação global do DNA foi realizada em subgrupo de 21 pacientes com restrição de crescimento e em 24 indivíduos controle. Dois tipos de análise foram realizados: (1) análise diferencial de grupo e (2) análise diferencial individual. Na primeira análise, em que foi comparado o padrão de metilação do grupo de pacientes com quadro clínico sugestivo de SRS (n=16) com o do grupo controle (n=24), não houve indicação de hipo ou hipermetilação global no grupo SRS. Na segunda análise, foi comparado o padrão de metilação de cada um dos 21 pacientes com restrição de crescimento e dos 24 indivíduos controle, com o padrão de metilação do grupo controle. O número médio de CpG hipermetilados e de segmentos diferencialmente metilados (SDM) foi significativamente maior nos pacientes. Foram identificados 82 SDM hipermetilados, estando 57 associados a gene(s) (69,5%), em 16 pacientes, e 51 SDM hipometilados, 41 deles associados a gene(s) (80,4%), em 10 pacientes. A análise de ontologia genética dos 61 genes associados aos SDM hipo ou hipermetilados nos pacientes destacou genes que atuam no desenvolvimento e na morfogênese do sistema esquelético e de órgãos fetais, e na regulação da transcrição gênica e de processos metabólicos. Alterações de metilação em genes que atuam em processos de proliferação e diferenciação celulares e crescimento foram identificadas em 9/20 dos pacientes (45%), sugerindo implicação clínica. Não foi detectada alteração epigenética comum aos pacientes com diagnóstico clínico de SRS, explicável provavelmente pela heterogeneidade clínica. A investigação de metilação global, utilizando microarray, produziu novos dados que podem contribuir para a compreensão de mecanismos moleculares que influenciam o crescimento pré- e pós-natal. Na translocação aparentemente equilibrada - t(5;6)(q35.2;p22.3)dn, detectada em paciente com suspeita clínica de SRS, a interrupção de um gene, pela quebra no cromossomo 6, pode ser a causa do quadro clínico; alternativamente, a translocação pode ter impactado a regulação de genes de desenvolvimento localizados próximos aos pontos de quebra. A análise de expressão em sangue periférico mostrou que os níveis de cDNA do gene, interrompido pelo ponto de quebra da translocação, estavam reduzidos à metade. Além de sinais típicos da SRS, a paciente apresentava algumas características clínicas sugestivas de displasia cleidocraniana. Assim, a translocação t(5;6) pode ter alterado a interação de genes de desenvolvimento e seus elementos reguladores, levando à desregulação de sua expressão espaço-temporal, e resultando num fenótipo atípico, com características sobrepostas de mais de uma síndrome genética / A large number of patients with growth disorders do not have the cause of their clinical phenotype established, including about 50% of patients with Silver-Russell syndrome (SRS), and 10-20% of patients with Beckwith-Wiedemann syndrome (BWS). The aim of this study was to investigate the (epi)genetic causes of growth disorders of unknown etiology, in a contribution to the understanding of growth regulation. The study included: (1) the investigation of submicroscopic chromosomal imbalances, by aCGH, (2) the analysis of the allele-specific expression profile of imprinted genes (IG), by pyrosequencing (PSQ) or Sanger sequencing, in patients with growth restriction; (3) the investigation of global methylation pattern in patients with growth restriction, using methylation microarray. The cohort consisted of 41 unrelated patients with growth disorders: (1) 25, with the diagnostic hypothesis of SRS; (2) six, with intrauterine growth restriction and birth weight below the 10th centile, associated with other clinical signs; (3) seven, with the diagnostic hypothesis of BWS; and (4) three, with prenatal or postnatal macrosomia, associated with other clinical signs. Chromosomal microdeletions and microduplications were investigated in 40 patients. Fifty-eight rare variants were detected in 30/40 patients (75%): 40 were considered likely benign (18 patients, 45%), 12, of unknown pathogenic significance (11 patients, 27.5%), two, likely pathogenic (one patient, 2.5%), and four, pathogenic (three patients, 7.5%). These frequencies are similar to those described in studies investigating CNVs in groups of patients with growth disorders and other congenital abnormalities, including SRS, and show the importance of investigating chromosomal microimbalances in these patients. The diversity of CNVs identified can be attributed to the clinical heterogeneity of these cohorts. In this study, many of the patients, with the diagnostic hypothesis of SRS or BWS, had atypical clinical signs, thus explaining the absence of specific SRS/BWS (epi)genetic mutations. The identification of CNVs, known to be causative of other syndromes, reflected the overlapping of some of their clinical features with those of SRS and BWS. The analysis of IG allele-specific expression profile was performed in a subgroup of 18 patients with growth restriction. Thirty IGs were initially selected, based on their association with cell proliferation, fetal growth or neurodevelopment. Transcribed SNPs with high frequency in the general population were selected for the genotyping of patients, parents and control subjects, determination of IG expression in peripheral blood, and of the monoallelic or biallelic expression pattern. The allele-specific expression of 13 IGs expressed in blood was then investigated in patients (seven of them by PSQ and six by Sanger sequencing). Expression alterations of two normally paternally expressed genes were detected in 4/18 patients (22%). This study is the first to use pyrosequencing and Sanger sequencing in the evaluation of IG allele-specific expression profile, in patients with growth restriction. Despite the limitations, both techniques have proved to be robust, and revealed interesting alterations in allelic expression; however, the causal relationship of these alterations with the clinical phenotypes remained unclear. The investigation of the global DNA methylation was performed in a subgroup of 21 patients with growth restriction, and in 24 control subjects. Two types of analysis were performed: (1) group differential analysis, and (2) individual differential analysis. In the first analysis, the methylation pattern obtained for the group of patients with the diagnostic hypothesis of SRS (n=16) was compared to that of the control group (n=24); no bias towards DNA hypo or hypermethylation was detected in the SRS group. In the second analysis, the methylation patterns of each of the 21 patients with growth restriction, and each of the 24 control subjects were compared to the methylation pattern of the control group. The average numbers of hypermethylated CpGs and of differentially methylated segments (DMSs) were significantly higher in the patients. In total, 82 hypermethylated DMSs - 57 associated with gene(s) (69.5%), in 16 patients, and 51 hypomethylated DMSs - 41 associated with gene(s) (80.4%), in 10 patients, were identified. Gene ontology analysis of the 61 DMS-associated genes highlighted genes involved in development and morphogenesis of the skeletal system and fetal organs, and also in the regulation of gene transcription and metabolic processes. Methylation changes in genes involved with cellular proliferation and differentiation, and growth were identified in 9/20 patients (45%), suggesting clinical implications; an epigenetic mutation common to SRS patients was not detected, likely due to the clinical heterogeneity of the cohort. The data generated by this global methylation analysis, using microarray, might contribute to the understanding of molecular mechanisms in growth restriction. In an apparently balanced translocation -t(5;6)(q35.2;p22.3)dn, detected in a patient with the diagnostic hypothesis of SRS, a gene found to be disrupted by the chromosome 6 breakpoint might explain the phenotype; alternatively, the translocation might have impacted the regulation of developmental genes in the vicinity of breakpoints. Expression analysis showed a significant decrease in the disrupted gene cDNA levels in the patient\'s blood cells, as expected. In addition to the SRS typical signs, the patient presented clinical features suggestive of cleidocranial dysplasia. Thus, the translocation t(5;6) might have altered the interaction of developmental genes and regulatory elements, leading to misregulation of spatiotemporal gene expression, thus resulting in an atypical phenotype, with overlapping features of more than one genetic syndrome

<div><div><div><p>Transcriptomic regulation of human liver is a tightly controlled and highly dynamic process. Genetic and environmental exposures to this process play pivotal roles in the development of multiple liver disorders. Despite accumulating knowledge have gained through large-scale genomics studies in the developed adult livers, the contributing factors to the interindividual variability in the pediatric livers remain largely uninvestigated. In the first two chapters of the present study, we addressed this question through an integrative analysis of both genetic variations and transcriptome-wide RNA expression profiles in a pediatric human liver cohort with different developmental stages ranging from embryonic to adulthood. Our systematic analysis revealed a transcriptome-wide transition from stem-cell-like to liver-specific profiles during the course of human liver development. Moreover, for the first time, we observed different genetic control of hepatic gene expression in different developmental stages. Motivated by the critical roles of genetics variations and development in regulating hepatic gene expression, we constructed robust predictive models to impute the virtual liver gene expression using easily available genotype and demographic information. Our model is promising in improving both PK/PD modeling and disease diagnosis for pediatric patients. In the last two chapters of the study, we analyzed the genomics data in a more liver disease- related context. Specifically, in the third chapter, we identified Macrophage migration inhibitory factor (MIF) and its related pathways as potential targets underlying human liver fibrosis through an integrative omics analysis. In the last chapter, utilizing the largest-to-date publicly available GWAS summary data, we dissected the causal relationships among three important and clinically related metabolic diseases: non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and obesity. Our analysis suggested new subtypes and provided insights into the precision treatment or prevention for the three complex diseases. Taken together, through integrative analysis of multiple levels of genomics information, we improved the current understanding of human liver development, the pathogenesis of liver disorders, and provided implications to precision medicine.</p></div></div></div>

Les cellules tumorales circulantes (CTCs) sont des cellules tumorales provenant de la tumeur primaire et/ou des métastases que l’on retrouve dans la circulation sanguine. Les plus agressives d’entre elles peuvent envahir les organes distants pour former des métastases. Leur faible nombre parmi la multitude de cellules sanguines rend difficile leurs détections et leurs études. C'est pourquoi, le challenge actuel est de pouvoir mettre en culture ces cellules.Dans le cadre de l’éude clinique nationale COLOSPOT, notre laboratoire a pu recueillir des échantillons de patients atteints d’un cancer colorectal métastatique. Grâce à ces prélèvements sanguins, 9 lignées dérivées de CTCs ont pu être établies : CTC-MCC-41, CTC-MCC-41.4, CTC-MCC-41.5A-G.Dans ce projet de thèse, les 9 lignées cellulaires ont été caractérisées au niveau du génome, du transcriptome, du protéome, du sécrétome et fonctionnel, et comparées à des lignées cellulaires tumorales primaires et métastatiques connues, comme effectuée précédemment sur la lignée CTC-MCC-41 (Cayrefourcq et al. 2015)Cette analyse très complète a montré malgré des profils génétiques très différents, toutes les lignées CTCs ont les caractéristiques d’un phénotype intermédiaire épithélial/mésenchymal, des propriétés de cellules souches, la mutation BRAFV600E et la capacité d’éviter divers processus de lutte contre les cellules tumorales comme la résistance à l’anoïkis et l’échappement au système immunitaire. Les études fonctionnelles ont montré que les CTC-MCC pouvaient induire rapidement la formation de tubes avec des cellules endothéliales in vitro, signe d'un potentiel angiogénique.La seconde partie de ce travail de thèse a été d’étudier la transition épithélio-mésenchymateuse (EMT) in vitro. Ce phénomène est une étape clé du processus métastatique des CTCs et implique diverses transformations des cellules à divers niveaux : morphologique, protéique et transcriptomique. Trois méthodes différentes ont été testées pour induire l’EMT au sein des lignées CTCs impliquant deux différents modes d’induction et deux modes de culture. Ces changements ont pu être observés dans les lignées témoins, validant les expérimentations effectuées. Cependant, l’EMT n’a pas été clairement observée sur les lignées CTCs.En conclusion, ces analyses suggèrent que les CTC coliques cultivés à partir de biopsies liquides séquentielles, effectuée durant le traitement d’un même patient, ont des caractéristiques communes. Mais la sélection des clones, avec un phénotype distinct, résistant au traitement, a été observée. D'autres études avec ces lignées CTC-MCC sont en cours, évaluant leur capacité à induire des tumeurs résistantes à des médicaments spécifiques ou à analyser la contribution épigénétique. Ces données peuvent fournir des indications pour la découverte de nouveaux biomarqueurs permettant d'identifier les sous-populations de CTC les plus agressives et pour la mise au point de nouveaux médicaments pour inhiber les CTCs initiatrices de métastases dans le cancer du côlon. / Circulating tumor cells (CTCs) are tumor cells that have been shed from the primary tumor and/or metastases into the bloodstream. The most aggressive ones can invade distant organs to form metastases. Their low number among the multitude of blood cells makes difficult their detection and study. This is why the current challenge in this field of expertise is to be able to culture them ex vivo.In the national COLOSPOT clinical study, our team was able to collect samples of patients with metastatic colorectal cancer. From blood samples of only one patient, 9 cancer cell lines derived from CTCs could be established: CTC-MCC-41, CTC-MCC-41.4, CTC-MCC-41.5A-G.In this project, the 9 CTC-MCC lines have been characterized at the genome, transcriptome, proteome, secretome and functional levels, and compared with primary and metastatic commercial colon cancer cell lines, as previously done on the CTC-MCC-41 line (Cayrefourcq et al., Cancer Res. 2015)These analyses have shown that despite their very different genetic profiles, all CTCs have the characteristics of an epithelial/mesenchymal intermediate phenotype, stemcell like characteristics, with BRAFV600E mutation, and the ability to avoid biological processes such as the resistance to anoïkis and the escape to the immune system. Moreover, functional studies have shown that all CTC-MCC lines can rapidly induce tubes formation with endothelial cells in vitro, a sign of an angiogenic potential.The second part of this thesis work was to study the epithelial-to-mesenchymal transition (EMT) in vitro. This phenomenon is a key step in the metastatic process and involves several cell transformations at various levels: morphological, proteomic and transcriptomic. Three different methods have been tested to induce EMT within these CTC-MCC lines involving two different induction and culture modes. These changes could be observed in the control lines, validating the experiments carried out. However, EMT has not been clearly observed yet on the CTC-MCC lines.In conclusion, this longitudinal study suggest that colorectal CTCs cultured from sequential liquid biopsies, performed during treatment of the same patient, have common characteristics. However, our results strongly suggest that no clonal selection, with a distinct phenotype, resistant to treatment, has occurred. Further studies with these CTC-MCC lineages are in process, evaluating their ability to induce in vivo drug-resistant tumors or to analyze the epigenetic contribution. These data may provide guidance for the discovery of new biomarkers to identify the most aggressive CTC subpopulations and for the development of novel drugs to inhibit metastases-competent CTCs in colon cancer.