Citopenia é um importante sinal de falência medular, sendo um achado comum de várias doenças, dentre as quais se destacam as mielodisplasias e a anemia aplástica. As mielodisplasias correspondem a um grupo de alterações hematopoéticas de natureza clonal, cuja principal característica é a hematopoese ineficaz, clinicamente manifesta como uma medula óssea celular, porém associada a citopenias. Já a anemia aplástica apresenta uma medula hipo ou acelular sem evidência de infiltração neoplásica, sendo substituída por tecido gorduroso. O gene GATA2 é um fator regulador da hematopoese, atuando também na manutenção e proliferação do pool de células-tronco e progenitoras hematopoéticas. Recentemente, mutações constitucionais no gene GATA2 foram descritas na síndrome de monocitopenia e infecção micobacteriana (MonoMac), que eventualmente cursa com outras citopenias, medula hipocelular ou mesmo mielodisplasia. Entretanto, a contribuição de mutações no gene GATA2 para o desenvolvimento de anemia aplástica adquirida e síndrome mielodisplásica não é conhecida. Neste trabalho, propomos pesquisar mutações no gene GATA2 em pacientes com anemia aplástica adquirida e síndrome mielodisplásica, por meio de sequenciamento direto do DNA. Adicionalmente, também avaliaremos as subpopulações linfocitárias no sangue periférico e níveis de citocinas plasmáticas no intuito de correlacionar a presença de mutação do GATA2 a um perfil imunológico. / Cytopenia is an important signal of marrow failure, being commom to various diseases, among them myelodysplasia and aplastic anemia. Myelodysplasia is a group of hematopoietic clonal disorders, with inneficient hematopoiesis, cellular bone marrow with associated cytopenias. The aplastic anemia presents a hypo or even acellular bone marrow without any evidence of neoplastic infiltration with the stem cells being substituted by fat. The GATA2 gene is a key regulator of hematopoiesis, also acting on the maintenance and proliferation of stem and progenitor cells. Recently, constitutional mutations in the GATA2 gene were described in MonoMAC syndrome, which eventually presents cytopenias, hypocellular marrow or even myelodysplasia. However, the contribution of GATA2 mutations for the development of acquired aplastic anemia or myelodysplasia is not known. In this work we aim to search for GATA2 gene mutations in patients with acquired aplastic anemia and myelodysplasia through Sanger sequencing. Also, we will evaluate the levels of subpopulations of lymphocytes and the plasmatic levels of cytokines to establish a correlation between the presence of mutation in the GATA2 and a specific immune profile

Citopenia é um importante sinal de falência medular, sendo um achado comum de várias doenças, dentre as quais se destacam as mielodisplasias e a anemia aplástica. As mielodisplasias correspondem a um grupo de alterações hematopoéticas de natureza clonal, cuja principal característica é a hematopoese ineficaz, clinicamente manifesta como uma medula óssea celular, porém associada a citopenias. Já a anemia aplástica apresenta uma medula hipo ou acelular sem evidência de infiltração neoplásica, sendo substituída por tecido gorduroso. O gene GATA2 é um fator regulador da hematopoese, atuando também na manutenção e proliferação do pool de células-tronco e progenitoras hematopoéticas. Recentemente, mutações constitucionais no gene GATA2 foram descritas na síndrome de monocitopenia e infecção micobacteriana (MonoMac), que eventualmente cursa com outras citopenias, medula hipocelular ou mesmo mielodisplasia. Entretanto, a contribuição de mutações no gene GATA2 para o desenvolvimento de anemia aplástica adquirida e síndrome mielodisplásica não é conhecida. Neste trabalho, propomos pesquisar mutações no gene GATA2 em pacientes com anemia aplástica adquirida e síndrome mielodisplásica, por meio de sequenciamento direto do DNA. Adicionalmente, também avaliaremos as subpopulações linfocitárias no sangue periférico e níveis de citocinas plasmáticas no intuito de correlacionar a presença de mutação do GATA2 a um perfil imunológico. / Cytopenia is an important signal of marrow failure, being commom to various diseases, among them myelodysplasia and aplastic anemia. Myelodysplasia is a group of hematopoietic clonal disorders, with inneficient hematopoiesis, cellular bone marrow with associated cytopenias. The aplastic anemia presents a hypo or even acellular bone marrow without any evidence of neoplastic infiltration with the stem cells being substituted by fat. The GATA2 gene is a key regulator of hematopoiesis, also acting on the maintenance and proliferation of stem and progenitor cells. Recently, constitutional mutations in the GATA2 gene were described in MonoMAC syndrome, which eventually presents cytopenias, hypocellular marrow or even myelodysplasia. However, the contribution of GATA2 mutations for the development of acquired aplastic anemia or myelodysplasia is not known. In this work we aim to search for GATA2 gene mutations in patients with acquired aplastic anemia and myelodysplasia through Sanger sequencing. Also, we will evaluate the levels of subpopulations of lymphocytes and the plasmatic levels of cytokines to establish a correlation between the presence of mutation in the GATA2 and a specific immune profile

L’importance des modificateurs de la chromatine dans la régulation de l’hématopoïèse et des hémopathies malignes est illustrée par l’histone méthyltransférase Mixed-Lineage Leukemia (MLL) qui est essentielle au maintien des cellules souches hématopoïétiques (CSH) et dont le gène correspondant, MLL, est réarrangé dans plus de 70% des leucémies du nourrisson. Les histones déméthylases (HDM), récemment découvertes, sont aussi impliquées dans le destin des CSH et des hémopathies malignes. Le but de ce projet est d’étudier l’expression des HDM dans les cellules hématopoïétiques normales et leucémiques afin d’identifier de potentiels régulateurs de leur destin. Nous avons réalisé un profil d'expression génique des HDM par qRT-PCR et par séquençage du transcriptome (RNA-seq) dans des cellules de sang de cordon (cellules CD34+ enrichies en CSH et cellules différenciées) et des cellules de leucémie aiguë myéloïde (LAM) avec réarrangement MLL. Les deux techniques montrent une expression différentielle des HDM entre les populations cellulaires. KDM5B et KDM1A sont surexprimés dans les cellules CD34+ par rapport aux cellules différenciées. De plus, KDM4A et PADI2 sont surexprimés dans les cellules leucémiques par rapport aux cellules normales. Des études fonctionnelles permettront de déterminer si la modulation de ces candidats peut être utilisée dans des stratégies d’expansion des CSH, ou comme cible thérapeutique anti-leucémique. Nous avons aussi développé et validé un nouveau test diagnostique pour détecter les mutations de GATA2 qui code pour un facteur de transcription clé de l’hématopoïèse impliqué dans les LAM. Ces travaux soulignent l’importance des facteurs nucléaires dans la régulation de l’hématopoïèse normale et leucémique. / The importance of chromatin modifiers in regulation of hematopoiesis and hematologic malignancies is illustrated by the Mixed-Lineage Leukemia (MLL) histone methyltransferase, which is essential to maintain hematopoietic stem cells (HSC) and whose corresponding gene, MLL, is rearranged in over 70% of infant leukemia. The recently discovered histone demethylases (HDM) are also involved in HSC fate and in hematologic malignancies. The purpose of this project is to study the expression of HDM in normal and leukemic hematopoietic cells to identify potential regulators of their fate. We performed a comprehensive gene expression profile of HDM by qRTPCR and transcriptome sequencing (RNA-seq) in cord blood cells (CD34+ cells enriched in HSC and differentiated cells) and in acute myeloid leukemia (AML) cells with MLL rearrangement. Both techniques revealed differential expression of HDM between these cell populations. KDM5B and KDM1A are overexpressed in CD34+ cells compared to differentiated cells. Moreover, KDM4A and PADI2 are overexpressed in leukemic cells compared to normal cells. Functional studies will determine whether modulation of these candidates can be used in HSC expansion strategies or as anti-leukemic drug target. We have also developed and validated a new diagnostic test to detect mutations of GATA2, a gene encoding a key transcription factor involved in hematopoiesis and in AML. This work highlights the importance of nuclear factors in the regulation of normal and leukemic hematopoiesis.