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1.
J Biol Chem ; 295(21): 7492-7500, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32299910

RESUMO

Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority of SCN cases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis.

2.
Blood Adv ; 4(6): 1131-1144, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32208489

RESUMO

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

4.
PLoS Genet ; 14(9): e1007642, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30216339

RESUMO

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.


Assuntos
Rim/metabolismo , Osmorregulação , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Células HEK293 , Humanos , Soluções Hipertônicas/farmacologia , Rim/efeitos dos fármacos , Masculino , Camundongos , Mutação , Osmorregulação/efeitos dos fármacos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Artigo em Inglês | MEDLINE | ID: mdl-29469136

RESUMO

BACKGROUND: Leri-Weill syndrome (LWS) ranks among conditions with short stature homeobox gene (SHOX) haploinsufficiency. Data on possible association of SHOX aberrations with malignant diseases are scarce. METHODS AND RESULTS: We report a unique case of an 8-year-old girl who was successfully treated for acute lymphoblastic leukemia (pre-B ALL, intermediate risk) and was subsequently diagnosed with LWS due to characteristic clinical appearance (short disproportionate stature, Madelung deformity of the wrist) and molecular genetic examination (complete deletion of SHOX). An identical SHOX deletion was identified also in the patient's mother. Leukemic cells of the patient were retrospectively examined by array comparative genomic hybridization (aCGH), which revealed five regions of deletions at chromosome X, including the SHOX gene locus. CONCLUSION: Growth retardation in children with hemato-oncologic malignancies cannot always be attributed to cytotoxic treatment and should be carefully evaluated, especially with regards to growth hormone therapy.


Assuntos
Deleção de Genes , Transtornos do Crescimento/complicações , Transtornos do Crescimento/genética , Osteocondrodisplasias/complicações , Osteocondrodisplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína de Homoeobox de Baixa Estatura/genética , Criança , Hibridização Genômica Comparativa , Feminino , Humanos , Perda de Seguimento , Linhagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico
6.
Semin Hematol ; 54(2): 81-86, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28637621

RESUMO

The GATA2 gene codes for a hematopoietic transcription factor that through its two zinc fingers (ZF) can occupy GATA-DNA motifs in a countless number of genes. It is crucial for the proliferation and maintenance of hematopoietic stem cells. During the past 5 years, germline heterozygous mutations in GATA2 were reported in several hundred patients with various phenotypes ranging from mild cytopenia to severe immunodeficiency involving B cells, natural killer cells, CD4+ cells, monocytes and dendritic cells (MonoMAC/DCML), and myeloid neoplasia. Some patients additionally show syndromic features such as congenital deafness and lymphedema (originally defining the Emberger syndrome) or pulmonary disease and vascular problems. The common clinical denominator in all reported cohorts is the propensity for myeloid neoplasia (myelodysplastic syndrome [MDS], myeloproliferative neoplasms [MPN], chronic myelomonocytic leukemia [CMML], acute myeloid leukemia [AML]) with an overall prevalence of approximately 75% and a median age of onset of roughly 20 years. Three major mutational types are encountered in GATA2-deficient patients: truncating mutations prior to ZF2, missense mutations within ZF2, and noncoding variants in the +9.5kb regulatory region of GATA2. Recurrent somatic lesions comprise monosomy 7 and trisomy 8 karyotypes and mutations in SETBP1 and ASXL1 genes. The high risk for progression to advanced myeloid neoplasia and life-threatening infectious complications guide decision-making towards timely stem cell transplantation.


Assuntos
Fator de Transcrição GATA2/deficiência , Predisposição Genética para Doença/genética , Neoplasias Hematológicas/genética , Transtornos Mieloproliferativos/genética , Fator de Transcrição GATA2/genética , Humanos
7.
Biochem Pharmacol ; 131: 52-67, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28193451

RESUMO

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.


Assuntos
Catepsina C/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Neutrófilos/enzimologia , Serina Proteases/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Estudos de Casos e Controles , Feminino , Humanos , Elastase de Leucócito/sangue , Macaca fascicularis , Doença de Papillon-Lefevre/enzimologia
8.
Blood ; 129(15): 2103-2110, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28179280

RESUMO

GATA family proteins play essential roles in development of many cell types, including hematopoietic, cardiac, and endodermal lineages. The first three factors, GATAs 1, 2, and 3, are essential for normal hematopoiesis, and their mutations are responsible for a variety of blood disorders. Acquired and inherited GATA1 mutations contribute to Diamond-Blackfan anemia, acute megakaryoblastic leukemia, transient myeloproliferative disorder, and a group of related congenital dyserythropoietic anemias with thrombocytopenia. Conversely, germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, whereas acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. The fact that mutations in these genes are commonly seen in blood disorders underscores their critical roles and highlights the need to develop targeted therapies for transcription factors. This review focuses on hematopoietic disorders that are associated with mutations in two prominent GATA family members, GATA1 and GATA2.


Assuntos
Fator de Transcrição GATA1 , Fator de Transcrição GATA2 , Doenças Hematológicas , Hematopoese , Mutação , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Animais , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Doenças Hematológicas/genética , Doenças Hematológicas/metabolismo , Humanos
9.
Cold Spring Harb Mol Case Stud ; 2(6): a001222, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27900365

RESUMO

Adult-onset Niemann-Pick disease type C (NPC) is an infrequent presentation of a rare neurovisceral lysosomal lipid storage disorder caused by autosomal recessive mutations in NPC1 (∼95%) or NPC2 (∼5%). Our patient was diagnosed at age 33 when he presented with a 10-yr history of difficulties in judgment, concentration, speech, and coordination. A history of transient neonatal jaundice and splenomegaly with bone marrow biopsy suggesting a lipid storage disorder pointed to NPC; biochemical ("variant" level cholesterol esterification) and ultrastructural studies in adulthood confirmed the diagnosis. Genetic testing revealed two different missense mutations in the NPC1 gene-V950M and N1156S. Symptoms progressed over >20 yr to severe ataxia and spasticity, dementia, and dysphagia with aspiration leading to death. Brain autopsy revealed mild atrophy of the cerebrum and cerebellum. Microscopic examination showed diffuse gray matter deposition of balloon neurons, mild white matter loss, extensive cerebellar Purkinje cell loss with numerous "empty baskets," and neurofibrillary tangles predominantly in the hippocampal formation and transentorhinal cortex. We performed whole-genome sequencing to examine whether the patient harbored variants outside of the NPC1 locus that could have contributed to his late-onset phenotype. We focused analysis on genetic modifiers in pathways related to lipid metabolism, longevity, and neurodegenerative disease. We identified no rare coding variants in any of the pathways examined nor was the patient enriched for genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) associated with longevity or altered lipid metabolism. In light of these findings, this case provides support for the V950M variant being sufficient for adult-onset NPC disease.


Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Sequência de Bases , Encéfalo/citologia , Encéfalo/patologia , Proteínas de Transporte/metabolismo , Demência/genética , Testes Genéticos , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Emaranhados Neurofibrilares/metabolismo , Doenças de Niemann-Pick/genética , Sequenciamento Completo do Genoma/métodos
10.
Science ; 353(6298): aaf7907, 2016 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-27229144

RESUMO

Multicellular systems develop from single cells through distinct lineages. However, current lineage-tracing approaches scale poorly to whole, complex organisms. Here, we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.


Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Linhagem da Célula , Rastreamento de Células/métodos , Endonucleases , Engenharia Genética/métodos , Animais , Proteína 9 Associada à CRISPR , Divisão Celular/genética , Código de Barras de DNA Taxonômico , Mutação , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/metabolismo , Peixe-Zebra , Zigoto
12.
J Clin Invest ; 125(8): 3103-16, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26193632

RESUMO

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPß-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.


Assuntos
Doenças Genéticas Inatas , Células-Tronco Pluripotentes Induzidas/enzimologia , Elastase de Leucócito , Mielopoese/genética , Neutropenia , Neutrófilos/enzimologia , Mutação Puntual , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático/genética , Feminino , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células Precursoras de Granulócitos/enzimologia , Humanos , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Masculino , Resposta a Proteínas não Dobradas/genética
13.
Br J Haematol ; 171(1): 13-28, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26018193

RESUMO

Lymphocytes are unique among cells in that they undergo programmed DNA breaks and translocations, but that special property predisposes them to chromosomal instability (CIN), a cardinal feature of neoplastic lymphoid cells that manifests as whole chromosome- or translocation-based aneuploidy. In several lymphoid malignancies translocations may be the defining or diagnostic markers of the diseases. CIN is a cornerstone of the mutational architecture supporting lymphoid neoplasia, though it is perhaps one of the least understood components of malignant transformation in terms of its molecular mechanisms. CIN is associated with prognosis and response to treatment, making it a key area for impacting treatment outcomes and predicting prognoses. Here we will review the types and mechanisms of CIN found in Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma and the lymphoid leukaemias, with emphasis placed on pathogenic mutations affecting DNA recombination, replication and repair; telomere function; and mitotic regulation of spindle attachment, centrosome function, and chromosomal segregation. We will discuss the means by which chromosome-level genetic aberrations may give rise to multiple pathogenic mutations required for carcinogenesis and conclude with a discussion of the clinical applications of CIN and aneuploidy to diagnosis, prognosis and therapy.


Assuntos
Instabilidade Cromossômica , Quebras de DNA , Replicação do DNA , DNA de Neoplasias , Neoplasias Hematológicas , Recombinação Genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/terapia , Humanos
14.
Nat Genet ; 47(2): 180-5, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25581430

RESUMO

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.


Assuntos
Neoplasias Hematológicas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Trombocitopenia/genética , Proliferação de Células , Éxons/genética , Feminino , Genes Reporter , Mutação em Linhagem Germinativa , Células HeLa , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Estrutura Terciária de Proteína , Proteínas Recombinantes , Análise de Sequência de RNA
15.
PLoS One ; 9(9): e106744, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25192356

RESUMO

Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.


Assuntos
Anatomia/educação , Educação Médica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Análise de Sequência de DNA/métodos , Cadáver , Grupo com Ancestrais do Continente Europeu/genética , Genoma Humano , Humanos , Fibrose Pulmonar Idiopática/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
16.
Blood ; 124(12): 1926-30, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25114263

RESUMO

Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.


Assuntos
Transtornos Herdados da Coagulação Sanguínea/genética , Transtornos Herdados da Coagulação Sanguínea/terapia , Transtornos Plaquetários/genética , Transtornos Plaquetários/terapia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação de Sentido Incorreto , Reparo Gênico Alvo-Dirigido/métodos , Animais , Transtornos Herdados da Coagulação Sanguínea/patologia , Transtornos Plaquetários/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/química , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/transplante , Leucemia Mieloide Aguda/patologia , Camundongos , Trombopoese/genética
17.
J Hematol Oncol ; 7: 36, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24754962

RESUMO

A 50-year-old woman was diagnosed with acute myeloid leukemia (AML). She has history of thrombocytopenia for 25 years and a significant family history of thrombocytopenia, affecting her mother, siblings and their children, as well as her own children. Both her mother and maternal aunt died from myelodysplastic syndrome (MDS). Additional genetic analysis was performed and identified two heterozygous missence mutations in the second zinc finger domain of GATA2 gene (p.Thr358Lys, and p.Leu359Val), occurring in cis on the same allele. Given the patient's family history and clinical manifestation, this was interpreted as an acute myeloid leukemia with heritable GATA2 mutations associated with familial AML-MDS. Germline GATA2 mutations are involved in a group of complex syndromes with overlapping clinical features of immune deficiency, lymphedema and propensity to acute myeloid leukemia or myelodysplastic syndrome (AML-MDS). Here we reported a case of familial AML-MDS with two novel GATA2 mutations. This case illustrates the importance of recognizing the clinical features for this rare category of AML-MDS and performing the appropriate molecular testing. The diagnosis of heritable gene mutations associated familial AML-MDS has significant clinical implication for the patients and affected families. Clinical trials are available to further investigate the role of allogeneic hematopoietic stem cell transplant in managing these patients.


Assuntos
Fator de Transcrição GATA2/genética , Leucemia Mieloide/genética , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/genética , Doença Aguda , Sequência de Bases , Análise Mutacional de DNA , Saúde da Família , Feminino , Heterozigoto , Humanos , Cariotipagem , Pessoa de Meia-Idade
19.
Blood ; 123(4): 562-9, 2014 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-24184683

RESUMO

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.


Assuntos
Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Mutação , Neutropenia/metabolismo , Biossíntese de Proteínas , Apoptose , Códon , Análise Mutacional de DNA , Retículo Endoplasmático/metabolismo , Células HL-60 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Neutrófilos/citologia , Fenótipo , Desnaturação Proteica , Dobramento de Proteína , Isoformas de Proteínas/metabolismo , Células U937
20.
Inflamm Bowel Dis ; 19(12): 2593-602, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24097228

RESUMO

BACKGROUND: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case-control study. METHODS: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics. RESULTS: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity. CONCLUSIONS: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields.


Assuntos
Células Clonais/patologia , Colite Ulcerativa/complicações , Neoplasias do Colo/etiologia , Lesões Pré-Cancerosas/etiologia , Telômero/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Estudos de Casos e Controles , Criança , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Neoplasias do Colo/patologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Poli G/genética , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/patologia , Prognóstico , Adulto Jovem
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