Estudamos os cariótipos de 50 espécimes (22 machos e 28 fêmeas) de Alouatta clamitans (bugio-ruivo) com técnicas citogenéticas tradicionais e de FISH com as sondas de pintura de todos os cromossomos humanos. Para os machos, foram observados dois números diploides diferentes (2n=45 e 49), com a ausência aparente do cromossomo Y devido à translocação Y-autossomo, e sete fórmulas cromossômicas distintas, com 17, 18, 19, 20, 21, 22 ou 24 cromossomos metacêntricos ou submetacêntricos e 21, 27, 28, 29, 30, 31 ou 32 cromossomos acrocêntricos. Para as fêmeas encontramos uma maior variabilidade no número diploide (2n=46, 48 e 50) e cinco fórmulas cromossômicas distintas, com 20, 22, 23 ou 25 cromossomos metacêntricos ou submetacêntricos e 21, 25, 27, 28 ou 30 cromossomos acrocêntricos. Os cromossomos X eram submetacêntricos, com exceção de duas fêmeas que apresentaram heteromorfismo neste par, com um dos cromossomos submetacêntrico e o outro metacêntrico. O sexo dos espécimes foi confirmado pela análise dos cariótipos. Pares heteromórficos autossomos também foram verificados. Dentre os indivíduos procedentes da Grande São Paulo, foram observadas as mesmas fórmulas cromossômicas em espécimes oriundos de diferentes fragmentos florestais, indicando que a fragmentação ainda não levou ao isolamento genético destas populações. A redução do número diploide orientada no sentido norte-;sul foi corroborada, com espécimes procedentes do estado de São Paulo apresentando 2n=49 ou 50 além de um exemplar do extremo sul deste estado com 2n=48 e os demais indivíduos oriundos de Santa Catarina com 2n=45 ou 46. Com a aplicação das técnicas de bandamento GTG e de FISH, foi possível verificar o sistema sexual múltiplo da espécie, do tipo X1X1X2X2X3X3/X1X2X3Y1Y2. Esta é a primeira descrição citogenética molecular com a hibridação de todas as sondas de cromossomos humanos em exemplares desta espécie com 2n=48, 49 e 50. Alguns cromossomos que apresentaram diferentes morfologias entre os espécimes analisados e foram responsáveis pelas diferentes fórmulas cromossômicas observadas, apresentaram regiões que não foram hibridadas por quaisquer sondas humanas. A partir da técnica de FISH foi possível determinar que o exemplar fêmea com 2n=48 é resultante do acasalamento de indivíduos portadores de diferentes cariótipos, com um dos genitores com o número diploide típico de espécimes procedentes da região sul (2n=45 ou 46) e o outro típico de espécimes procedentes da região sudeste do Brasil (2n= 49 ou 50). Este padrão levaria à formação de dois trivalentes durante a divisão meiótica, com implicações causadas para a formação dos gametas que poderiam reduzir ou impedir a fertilidade dos indivíduos portadores deste cariótipo, constituindo um mecanismo de isolamento pós−zigótico e indicando que as populações do sudeste e sul do Brasil já estão isoladas a ponto de constituírem espécies diferentes. Também analisamos com citogenética tradicional e com a hibridação de todas as sondas de cromossomos humanos um exemplar que foi apreendido pelo IBAMA e entregue ao DEPAVE-3 e que apresentou características morfológicas divergentes das encontradas para Alouatta clamitans. Os dados nos levaram a concluir que este indivíduo é um representante de Alouatta ululata, sendo esta a primeira descrição cariotípica desta espécie, restrita geograficamente ao norte do estado do Maranhão, Piauí e Ceará. Sendo assim, a citogenética se mostrou uma importante ferramenta para a identificação da espécie e da correta origem geográfica dos indivíduos, além de ter contribuído para evitar a introdução na fauna do município de São Paulo de um exemplar não endêmico desta região. Contribuímos também para a reintrodução de outros indivíduos que fizeram parte do Projeto “Manejo e Conservação do Bugio, Alouatta clamitans (Primates, Atelidae) na Região Metropolitana de São Paulo: aprimorando o programa de reintrodução”, em parceria com o DEPAVE-3. / We studied the karyotypes of 50 specimens (22 males and 28 females) of Alouatta clamitans (brown howler monkey) with traditional and FISH cytogenetic techniques with painting probes of all human chromosomes. For the males were observed two different diploid number (2n=45 and 49), with the apparent absence of Y chromosome due to translocation Y-autosome and seven distinct chromosomal formulas, with 17, 18, 19, 20, 21, 22 or 24 biarmed chromosomes and 21, 27, 28, 29, 30, 31 or 32 acrocentric chromosomes. For females we found greater variability in the diploid number (2n = 46, 48 and 50) and five distinct chromosomal formulas, 20, 22, 23 or 25 biarmed chromosomes and 21, 25, 27, 28 or 30 acrocentric chromosomes. The X chromosomes were submetacentric, except for two females who had heteromorphic pairs, with one of submetacentric chromosomes and the other metacentric. Autosomes heteromorphic pairs were also observed. Among the coming individuals in the Grande São Paulo, the same chromosomal formulas in specimens from different forest fragments were observed, indicating that fragmentation has not yet led to genetic isolation these populations. The reductions of diploid number oriented in north−south direction was observed, with coming state specimens from São Paulo presented 2n=49 or 50 as well as an extreme Southern copy of this state with 2n=48 and other individuals from Santa Catarina with 2n=45 or 46. We verified the multiple sex chromosome system X1X1X2X2X3X3/X1X2X3Y1Y2. This is the first description of hybridization with all human chromosomes painting probes in specimens with 2n=48, 49 and 50. It was also determined that the female specimen with 2n=48 is the result of the mating of individuals with different karyotypes, with one parent with the typical diploid number of specimens coming from the South (2n=45 or 46) and other typical specimens coming from the Southeast of Brazil (2n=49 or 50). This standard would lead to the formation of two trivalent during meiotic division, with implications due to the formation of gametes that could reduce or prevent the fertility of individuals of this karyotype, being a post−zygotic isolation mechanism and indicating that the southeastern and southern Brazil populations are already isolated enough to constitute different species. We also analyze a specimen that was seized by IBAMA and delivered to DEPAVE-3 and presented different morphological characteristics found to Alouatta clamitans, the species occurring in the supposed geographic region origin of this specimen (Ibiúna/SP). This individual was classified as Alouatta ululata, geographically restricted to the northern state of Maranhão, Piauí and Ceará. Thus, karyological studies proved an important tool for species identification and the correct geographic origin of individuals. We also contributed to reintroducing individuals who were part of the project “Manejo e Conservação do bugio Alouatta clamitans (Primates, Atelidae) na Região Metropolitana de São Paulo: aprimorando o programa de reintrodução” in partnership with the DEPAVE−3.

A displasia frontonasal (DFN) compreende quadros de aparência facial variável, sendo clinicamente caracterizada por dois ou mais dos seguintes sinais: hipertelorismo ocular com consequente alargamento da base nasal; fissura facial mediana afetando o nariz ou o nariz e lábio superior e, por vezes, o palato; fissura alar (uni ou bilateral); ponta nasal ausente; crânio anterior bífido oculto, e implantação em 'V' dos cabelos na fronte. A DFN pode ser vista como um defeito de desenvolvimento que pode ocorrer por si só ou como parte do quadro clínico de várias síndromes. A maioria dos casos de DFN é esporádica, e em raras circunstâncias foram observadas alterações cromossômicas em alguns indivíduos. Até o momento, quatro genes foram relacionados à patogênese molecular de algumas das síndromes com DFN, EFNB1, associado a uma forma de DFN ligada ao X e os genes ALX1, ALX3 e ALX4, todos associados a formas de DFN com herança autossômica recessiva. Embora esteja claro haver heterogeneidade etiológica, na maioria dos casos de DFN a causa não é conhecida, dificultando o adequado aconselhamento genético aos pacientes e seus familiares. Sendo assim, realizamos estudos com diferentes estratégias metodológicas buscando melhor compreender as possíveis causas genéticas da DFN. Ao todo foram analisados 10 pacientes: um caso familial de DFN leve com herança aparentemente autossômica dominante, um caso clinicamente sugestivo de mutação em ALX1, e oito casos de DFN associada a atraso de desenvolvimento com ou sem outras anomalias, dos quais um apresentava um rearranjo de novo aparentemente balanceado entre os cromossomos 4 e 12. Optamos por realizar sequenciamento dos genes previamente relacionados a fenótipos com DFN em todos os casos; para aqueles em que não foram detectadas mutações patogênicas, realizamos análise de variações de número de cópias (CNV) por microarray de polimorfismos de base única e, para o paciente com rearranjo cromossômico, realizamos o mapeamento do ponto de quebra por hibridação in situ fluorescente. Constatamos uma mutação em heterozigose no gene ALX4 co-segregando com o fenótipo do caso familial, sendo esta a primeira descrição de alteração em tal gene causando uma forma de DFN com herança dominante, e sugerimos pela primeira vez um mecanismo de dominância negativa. No caso sugestivo de mutação em ALX1, o diagnóstico foi confirmado através da identificação de uma mutação em homozigose neste gene do paciente; este caso consiste no 3o da literatura mundial e evidencia pela primeira vez que mutações em ALX1 não necessariamente levam a atraso de desenvolvimento ou deficiência intelectual. Os estudos citogenéticos e moleculares dos pontos de quebra do paciente com rearranjo cromossômico sugeriram os genes ARAP2 e CAND1 como possíveis responsáveis por seu quadro clínico, enquanto o estudo de CNVs nos indivíduos com DFN associada a atraso de desenvolvimento apontou os genes DNAJB12 e ENOX2 como possíveis candidatos para explicar o fenótipo de dois dos pacientes. É preciso que novos estudos sejam realizados a fim de melhor compreender o significado de tais achados e a real contribuição de cada gene para o desenvolvimento craniofacial humano e para a etiologia da DFN. Para os casos em que não foram identificadas alterações conclusivas no presente estudo, embora causas ambientais não possam ser descartadas, é preciso que seja investigada também a existência de fatores genéticos e epigenéticos não detectáveis pelas metodologias utilizadas, bem como a hipótese de mosaicismo somático. Nossos resultados, além de corroborarem o envolvimento dos genes ALX1 e ALX4 em fenótipos com DFN, sugerem também novos genes candidatos: ARAP2, CAND1, DNAJB12 e ENOX2 / Frontonasal dysplasia (FND) is a rare group of disorders that comprises cases with a variety of facial appearances, and is clinically characterized by two or more of the following signs: ocular hypertelorism with consequent broadening of the nasal root; median facial cleft affecting the nose and/or upper lip and palate; clefting of the alae nasi (uni or bilateral); lack of formation of the nasal tip; anterior cranium bifidum occultum; and a V-shaped frontal hairline. FND is a developmental defect that can occur alone or as part of several syndromes. Most cases of FND are sporadic, and in rare circumstances chromosomal alterations were observed in affected individuals. To date, four genes have been related to the molecular pathogenesis of some syndromes with DFN, one (EFNB1) is associated with an X-linked form while the 3 others (ALX1, ALX3 and ALX4) are associated with autosomal recessive forms. Although it is clear that FND is etiologic heterogeneous, the causative mechanism is unknown in most cases which makes it hard to give proper genetic counseling to patients and their families. In order to get new insights into the genetic mechanisms leading to FND, we performed studies with different methodologies. Altogether, 10 patients were analyzed: a familial case of a mild form of FND with an apparently autosomal dominant inheritance pattern, a case clinically suggestive of mutation in ALX1, and eight cases of FND associated with developmental delay with or without other anomalies, one of which with an apparently balanced de novo rearrangement between chromosomes 4 and 12. We chose to sequence the genes previously associated with FND phenotypes in all cases; for those in which pathogenic mutations were not detected, we conducted an analysis of copy number variations (CNV) by single nucleotide polymorphisms microarrays; for the patient with chromosomal rearrangement, we also mapped the breakpoints by using fluorescence in situ hybridization. We found a heterozygous mutation in ALX4 co-segregating with the phenotype of the familial case; this is the first description of mutation in this gene causing a form of FND with dominant inheritance pattern, and we suggested for the first time a dominant negative mechanism. In the case suggestive of mutation in ALX1, the diagnosis was confirmed by the identification of a homozygous mutation in this gene; this is the third case of the literature and shows for the first time that mutations in ALX1 are not necessarily related to developmental delay or intellectual disability. Breakpoints cytogenetic and molecular studies done with the patient with chromosomal rearrangement suggested ARAP2 and CAND1 genes as causative candidates for his condition, while the study of CNVs in individuals with FND associated with developmental delay pointed DNAJB12 and ENOX2 genes as possible candidates to explain the phenotypes of two of the patients. Further studies are necessary to better understand the significance of such findings and the actual contribution of each of these genes to human craniofacial development and the etiology of FND. Although environmental causes cannot be ruled out, it should also be investigated the existence of genetic and epigenetic factors as well as the possibility of somatic mosaicism, among the cases negative for the molecular approaches used in our study. Our results corroborate the involvement of ALX1 and ALX4 in FND phenotypes, and suggest new candidate genes: ARAP2, CAND1, DNAJB12 and ENOX2.

No description available.

DuMPLING is a newly developed high-throughput method to study singlecellphenotypes in a pooled and barcoded library using a microfluidicchip. The chip enables parallel biophysical measurements of singlecells, after which in-situ genotyping connects the cells to a certainstrain of the library. The method has been previously applied with abarcoded library, where genotyping was performed on barcodes presenton high copy number plasmids. In this project, I apply and developthe Rolling Circle Amplification method to amplify the signal frombarcodes present on the E. coli chromosome. A small librarycontaining three different chromosomal barcodes is investigated. Veryhigh efficiency of signal generation is achieved for the firstbarcode, good efficiency is achieved for the second, and no signal isachieved for the third. Genotyping is also successfully performed ona strain with two different barcodes present on the chromosome. Thegenotyping method described herein can be applied to screen foradditional barcodes that may be incorporated in a larger library thatin turn can be used to ask important biological questions, forexample using the high throughput DuMPLING method.

Les translocations chromosomiques sont associées à un grand nombre de cancers. Les translocations chromosomiques sont impliquées dans la tumorigenèse par différents mécanismes : elles conduisent soit à une dérégulation d’un oncogène, soit à la formation d’un nouvel oncogène de fusion. Cependant, le lien direct entre l'apparition d'une translocation chromosomique et la formation d'une tumeur n'est pas totalement établi. Par exemple, plusieurs translocations associées au cancer ont été détectées dans le sang d’individus sains voire dans le sang de cordon des bébés avec une prévalence bien supérieure à celle de la maladie. Ceci suggère que la seule formation de la translocation ne suffit pas toujours à induire l’oncogenèse. La plupart des travaux de recherche antérieurs reposaient sur la surexpression de la protéine de fusion, oncogène supposé. Ces approches présentent de nombreuses limites, la translocation chromosomique est alors absente de même que le contexte chromosomique natif du gène de fusion (promoteur endogène, statut de la chromatine, etc.) ou les éventuels effets d’haplo-insuffisance qui ne sont pas récapitulés. La molécule d’ADN étant organisée de manière non aléatoire dans le noyau, les réarrangements chromosomiques sont également susceptibles d’affecter le statut épigénétique, la réplication et la transcription du chromosome dérivatif entier, en plus des segments d’ADN nouvellement juxtaposés. Or la technologie CRISPR/Cas9, permet de reproduire la translocation chromosomique in situ, après avoir induit deux cassures double-brin simultanées. Ce travail de thèse a porté spécifiquement sur la translocation t(2,5) (p23, q35) qui induit l’expression de la protéine de fusion NPM1-ALK fréquemment rencontrée dans le lymphome anaplasique à grandes cellules (ALCL). Nous avons reproduit la t(2,5) à la fois dans des lignées cellulaires mais aussi dans des cellules T primaires à la fin de ma thèse. Nous avons pu montrer des modifications significatives du timing de réplication des cellules qui portent la translocation en comparaison des cellules isogéniques de départ (par la méthode du Répli-seq) pouvant avoir un impact sur l’homéostasie des cellules tumorales. En parallèle, nous avons mis en évidence la formation d'ARN circulaires de fusion spécifiques, exprimés à partir du gène de fusion, spécifiques des lignées tumorales. Ces ARN circulaires pourraient donner naissance à de nouveaux biomarqueurs diagnostic/pronostic dans le futur. Ces travaux permettront de mieux comprendre les conséquences des translocations chromosomiques oncogéniques dans les cellules humaines et pourraient mener vers de nouvelles orientations thérapeutiques à l’avenir. / Chromosomal translocations are associated with a wide range of cancers. These chromosomal rearrangements are implicated in tumorigenesis by different mechanisms: either they lead to oncogene upregulation or tumor suppressor downregulation. However, the direct link between the appearance of one chromosomal translocation and tumor formation is not always clear. For example, several cancer translocations have been found in PBMCs or in cord blood cells from healthy individuals, suggesting that translocation formation alone is not always sufficient to drive oncogenesis. Most of previous research works on cancer translocation relied on studies using overexpression of the fusion protein. These approaches do not reproduce the chromosome arm translocation nor the chromosomal context of the fusion gene (endogenous promotor, chromatin status etc…) or do not recapitulate a potential haplo-insufficiency of the translocated cells. Because the DNA molecule is organized non-randomly in the nucleus, chromosomal rearrangements are also likely to impact the epigenetic, replication and transcriptional status of the whole rearranged chromosome in addition to the newly juxtaposed gene segments. Using CRISPR/Cas9 technology, we can recapitulate chromosomal translocation in situ, after inducing 2 concurrent double-strand breaks. In this work, we focus on t(2,5)(p23,q35) leading to NPM1-ALK fusion protein frequently found in Anaplasic Large Cell Lymphoma (ALCL). We could recapitulate t(2;5) in cell lines but more importantly in human primary T cells from healthy donors. We showed significant modifications on Replication Timing in model cell lines compare to isogenic non-translocated cells (using Repli-seq analysis). Importantly, these changes might have a direct impact on tumor cell homeostasis. In parallel, we also highlighted the formation of specific fusion circular RNAs expressed from the fusion gene also found in tumor cells. These circular RNAs could give rise to new diagnostic/prognostic biomarkers in the future. This work will lead to a better understanding of the consequences of cancer translocation in human cells and could give new directions for therapeutics in future.

No description available.

Thesis (PhD (Psychiatry))--Stellenbosch University, 2008. / BACKGROUND: Schizophrenia is a heterogeneous illness resulting from complex gene-environment interplay. The majority of molecular genetic work done has involved Caucasian populations, with studies in these and Asian populations showing 2-32% of sufferers to have chromosomal aberrations. So far the discovery of a specific susceptibility mechanism or gene still eludes us, but the use of endophenotypes is advocated as a useful tool in this search. No cytogenetic studies of this nature have been reported in any African schizophrenia population. AIM: The aim of the study was to combine genotypic and phenotypic data, collected in a homogenous population in a structured manner, with the hope of characterising an endophenotype that could be used for more accurate identification of individuals with possible chromosomal abnormalities. METHODOLOGY: A structured clinical interview was conducted on 112 Xhosa schizophrenia patients. (Diagnostic Interview for Genetic Studies, including Schedules for the Assessment of Negative and Positive Symptoms.) Blood samples (karyotyping and/or FISH analysis) as well as urine samples (drug screening) were obtained and nine head and facial measurements were performed. Descriptive statistics were compiled with reference to demographic, clinical and morphological variables. Comparisons between mean differences for these variables were made.

Récemment, une nouvelle classe d'ADN circulaire extrachromosomique (eccDNA) appelée microADN a été identifiée dans des tissus humains et murins. Ces microADNs ont une longueur de 100 à 400 pb, sont dérivés de régions génomiques non répétitives uniques et présentent un enrichissement au niveau des régions géniques et riches en GC. Bien qu'il ait été proposé qu'ils puissent provenir du métabolisme de l'ARN ou des défauts de réplication, leurs mécanismes de production et leur éventuelle fonctionnalité restent à déterminer. Grâce à l'analyse des microADNs extraits d'une série de 10 lignées cellulaires lymphoblastoïdes humaines (LCL), nous avons confirmé la distribution nonaléatoire des microADNs vers les régions actives du génome. Les microADNs identifiés présentaient des loci d'origine redondants et une périodicité de taille de 190 pb pouvant correspondre à la fragmentation de l'ADN lors de l'apoptose caspase-dépendante. L'apoptose induite de ces LCLs par des drogues chimiothérapeutiques (méthotrexate ou L-asparaginase) a entrainé la modulation de la diversité et de la taille des microADNs, suggérant qu'une partie de ces entités pourrait être des produits résiduels de la mort cellulaire apoptotique. Ainsi, bien que compatible avec l'observation initiale suggérant que les microADNs proviennent d'un processus physiologique normal, ces résultats impliquent une source de production alternative ou complémentaire. / Recently, a new class of extrachromosomal circular DNA (eccDNA) called microDNA was identified in mouse and human tissues. These microDNAs are 100 to 400 bp long, derive from unique nonrepetitive genomic regions and show an enrichment in GC rich and genic sequences. While it has been proposed that they could arise from RNA metabolism or replication defects, their production mechanisms and eventual functionality remain unclear. Through the analysis of microDNAs extracted from a series of 10 human lymphoblastoid cell lines (LCLs), we confirmed the non-random distribution of microDNA towards active regions of the genome. Identified microDNAs showed redundant loci of origin and a size periodicity of 190 bp that matched caspase-dependant DNA fragmentation of apoptotic cells. Strikingly, the chemotherapeutic drug-induced apoptosis (using methotrexate or Lasparaginase) of these LCLs modulated both diversity and size of microDNAs further suggesting that a part of microDNAs could represent circularized by-products of the programmed cell death. Thus, while compatible with the original observation that microDNAs originated from a normal physiological process, these results imply an alternative or complementary source of production.

La leucémie myéloïde chronique (LMC) est un modèle d’évolution tumorale dans les cancers humains. Le processus d’évolution de la LMC de la phase chronique (PC) à la phase blastique (PB) est caractérisé par un arrêt de différenciation et l’acquisition de la capacité d’autorenouvellement incontrôlé d’une cellule souche ou d’un progéniteur hématopoïétique. La LMC en PB est associée à la présence d’anomalies génétiques additionnelles à la fusion BCR-ABL1 qui résulte de la translocation chromosomique t(9;22). Contrairement aux patients en PC, les patients en PB de la LMC n’obtiennent pas une réponse moléculaire complète à long terme avec 1’Imatinib mesylate, un inhibiteur de la tyrosine kinase (ITK) BCR-ABL1. De plus, les ITKs de deuxième et troisième générations sont moins efficaces en PB de la LMC lorsque les cellules leucémiques ont acquis une résistance au traitement indépendante des mutations de BCR-ABL1. Les mécanismes moléculaires des voies de signalisation impliquées dans la progression de la LMC en PB ne sont pas entièrement élucidés. Le but de notre travail est de caractériser de nouvelles anomalies génétiques dans la PB de la LMC. Nous avons identifié en cytogénétique, quatre nouvelles translocations chromosomiques : t(1;21)(p36;q22), t(7;17)(p15;q22), t(8;17)(q11;q22) et t(2;12)(q31;p13) dans les cellules leucémiques de patients en PB de la LMC résistants au traitement. En utilisant des techniques d'hybridation in situ en fluorescence, de RT-PCR et de séquençage, nous avons délimité les régions à investiguer au niveau des points de cassure et identifié un réarrangement de plusieurs gènes codant pour des facteurs de transcription importants lors de l’hématopoïèse tels que RUNX1, ETV6, PRDM16 et HOXA. L’altération de ces gènes pourrait expliquer l’arrêt de différenciation et/ou l’acquisition de la capacité d’autorenouvellement caractéristiques de la LMC en PB. Nous avons identifié les fusions RUNX1-PRDM16, MSI2-HOXA, MSI2-SOX17 et ETV6-HOXD11, respectivement associées aux translocations chromosomiques t(1;21), t(7;17), t(8;17) et t(2;12). Ces fusions génèrent différents transcrits alternatifs qui maintiennent et altèrent le cadre ouvert de lecture. L’analyse des séquences des transcrits chimériques identifiés dans ce projet, incluant RUNX1-PRDM16, MSI2-HOXA9, MSI2-HOXA10, MSI2-HOXA11 et ETV6-HOXD11, nous a permis de prédire les domaines fonctionnels potentiellement présents au niveau des protéines chimériques prédites. Les transcrits de fusion qui respectent le cadre ouvert de lecture peuvent générer des domaines fonctionnels des deux partenaires. C’est le cas des deux transcrits identifiés pour la fusion RUNX1-PRDM16 où le domaine de liaison à l’ADN RHD (Runt homology domain) de RUNX1 est fusionné avec la quasi-totalité des domaines de PRDM16. Les transcrits de fusion qui ne respectent pas le cadre ouvert de lecture donnent des formes tronquées des transcrits RUNX1, MSI2 et ETV6. La juxtaposition des régions promotrices de ces derniers en 5’ de leurs partenaires entraîne l’activation de la forme courte oncogénique de PRDM16 dans la t(1;21) ou de différents gènes HOXA/D dans les t(7;17) et t(2;12), ainsi que l’expression aberrante d’un nouveau transcrit alternatif de SOX17 dans la t(8;17). Notre étude nous a permis d’identifier de nouveaux gènes de fusion et/ou une activation de gènes qui pourraient coopérer avec la fusion BCR-ABL1 dans la progression de la LMC et être impliqués dans la résistance au traitement de la LMC en phase avancée. La caractérisation des événements génétiques associés à la transformation blastique de la LMC est essentielle pour l’investigation des voies moléculaires impliquées dans cette phase de la maladie. Investiguer la résistance au traitement de ces patients pourrait aussi contribuer à identifier de nouvelles cibles thérapeutiques dans cette leucémie. / Chronic myeloid leukemia (CML) is a model of tumor evolution in human cancer. The evolution process of CML from the chronic phase (CP) to the blastic phase (BP) is characterized by a blockade of differentiation and acquisition of uncontrolled self-renewal capacity by hematopoietic stem or progenitor cells. CML-BP is associated with the presence of other genetic abnormalities in addition to the BCR-ABL1 fusion which results from chromosomal translocation t(9;22). Unlike patients in the CP, patients with CML-BP do not achieve a long-term complete molecular response to Imatinib mesylate, an inhibitor targeting the BCR-ABL1 tyrosine kinase (TK). Moreover, second and third generation TK inhibitors are less effective in CML-BP when leukemic cells have acquired a therapeutic resistance independent of BCR-ABL1 mutations. The molecular mechanisms of the signaling pathways responsible for CML progression from CP to BP are poorly understood. The aim of our project is to characterize novel genetic alterations in the BP of CML. We have identified by cytogenetics, four novel chromosomal translocations: t(1;21)(p36;q22), t(7;17)(p15;q22), t(8;17)(q11;q22) and t(2;12)(q31;p13) in leukemic cells of patients with CML-BP resistant to therapy. Using fluorescence in situ hybridization, RT-PCR and sequencing techniques, we have mapped chromosomal translocation breakpoints and identified rearranged genes encoding transcription factors which are key regulators of hematopoiesis, such as RUNX1, ETV6, PRDM16 and HOXA. The disruption of these genes could explain the differentiation blockade and/or uncontrolled self-renewal associated with the CML-BP. We identified RUNX1-PRDM16, MSI2-HOXA, MSI2-SOX17 and ETV6-HOXD11 fusions created by chromosomal translocations t(1;21), t(7;17), t(8;17) and t(2;12) respectively. These fusions generate different alternative transcripts that both maintain and alter the open reading frame. Sequence analysis of chimeric transcripts identified in this project, including RUNX1-PRDM16, MSI2-HOXA9, MSI2-HOXA10, MSI2-HOXA11 and ETV6-HOXD11, allowed us to predict potential functional domains present in putative chimeric proteins. In-frame fusion transcripts can generate functional domains from both fusion partners. For example, in two RUNX1-PRDM16 transcripts, the RUNX1 DNA binding domain RHD (Runt homology domain) is fused to the majority of PRDM16 domains. Out-of-frame fusion transcripts resulted in truncated forms of RUNX1, MSI2 and ETV6. The juxtaposition of promoter regions of these genes to the 5’ part of their partners resulted in the activation of the oncogenic short form of PRDM16 in the t(1;21) or of different HOXA/D genes in t(7;17) and t(2;12), and in the aberrant expression of a novel alternative SOX17 transcript in the t(8;17). Our study allowed us to identify novel fusion genes and/or activation of genes that potentially cooperate with BCR-ABL1 fusion in the progression of CML and contribute to treatment resistance of this disease. The characterization of genetic events related to the blastic transformation of CML is an important step in the investigation of molecular pathways involved in this stage of the disease. Understanding treatment resistance of these patients might help to identify new therapeutic targets in this leukemia.

Afin d’effectuer des études fonctionnelles sur le génome de la souris, notre laboratoire a généré une bibliothèque de clones de cellules souches embryonnaires (ESC) présentant des suppressions chromosomiques chevauchantes aléatoires – la bibliothèque DELES. Cette bibliothèque contient des délétions couvrant environ 25% du génome murin. Dans le laboratoire, nous comptons identifier de nouveaux déterminants du destin des cellules hématopoïétiques en utilisant cet outil. Un crible primaire utilisant la benzidine pour démontrer la présence d'hémoglobine dans des corps embryoïdes (EBS) a permis d’identifier plusieurs clones délétés présentant un phénotype hématopoïétique anormal. Comme cet essai ne vérifie que la présence d'hémoglobine, le but de mon projet est d'établir un essai in vitro de différenciation des ESC permettant de mesurer le potentiel hématopoïétique de clones DELES. Mon hypothèse est que l’essai de différenciation hématopoïétique publié par le Dr Keller peut être importé dans notre laboratoire et utilisé pour étudier l'engagement hématopoïétique des clones DELES. À l’aide d’essais de RT-QPCR et de FACS, j’ai pu contrôler la cinétique de différenciation hématopoïétique en suivant l’expression des gènes hématopoïétiques et des marqueurs de surface comme CD41, c-kit, RUNX1, GATA2, CD45, β-globine 1 et TER-119. Cet essai sera utilisé pour valider le potentiel hématopoïétique des clones DELES candidats identifiés dans le crible principal. Mon projet secondaire vise à utiliser la même stratégie rétro-virale a base de Cre-loxP utilisée pour générer la bibliothèque DELES pour générer une bibliothèque de cellules KBM-7 contenant des suppressions chromosomiques chevauchantes. Mon but ici est de tester si la lignée cellulaire leuémique humaine presque haploïde KBM-7 peut être exploitée en utilisant l'approche DELES pour créer cette bibliothèque. La bibliothèque de clones KBM-7 servira à définir les activités moléculaires de drogues anti-leucémiques potentielless que nous avons identifiées dans le laboratoire parce qu’elles inhibent la croissance cellulaire dans plusieurs échantillons de leucémie myéloïde aiguë dérivés de patients. Elle me permettra également d'identifier les voies de signalisation moléculaires qui, lorsque génétiquement perturbées, peuvent conférer une résistance à ces drogues. / To carry out functional studies on the mouse genome, our laboratory has generated a library of Embryonic Stem Cell (ESC) clones harboring random nested chromosomal deletions – DELES library. This library contains deletions covering ~ 25% of the mouse genome. In the lab, we are interested in identifying novel hematopoietic cell fate determinants using this resource. A primary screen using benzidine to demonstrate the presence of hemoglobin in embryoid bodies (EBs) was able to identify several DELES clones exhibiting abnormal hematopoietic phenotype. Since this assay only tested for the presence of hemoglobin, the goal of my project is to establish a robust in vitro ESC differentiation assay to monitor the hematopoietic potential of DELES clones. My hypothesis is that the hematopoietic differentiation assay published by Dr. Keller can be used to observe hematopoietic commitment of the DELES clones. Using QRT-PCR and FACS assays I was able to monitor the kinetics of hematopoietic differentiation by observing the expression of hematopoietic genes and surface markers including CD41, C-KIT, RUNX1, GATA2, CD45, β-GLOBIN 1 and TER-119. This assay will be used to validate the hematopoietic potential of the candidate DELES clones identified in the primary screen. My secondary project aims to use the same retro-viral Cre-loxP strategy used for the DELES library, in order to generate a library of KBM-7 leukemic cells harboring nested chromosomal deletions. My goal here is to test if the human near haploid KBM-7 cell line can be exploited using the DELES approach to create this library. The library of KBM-7 clones will be used to delineate the molecular activities of potential anti-leukemic drugs that we have identified in the lab to inhibit cell growth in several patient-derived acute-myeloid leukemia specimens. It will also allow me to identify molecular signaling pathways that, when genetically disrupted, can confer resistance to these drugs.