RESUMEN
The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.
Asunto(s)
Factor de Unión a CCAAT/genética , Diferenciación Celular/genética , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Isoformas de Proteínas/genética , Animales , Factor de Unión a CCAAT/biosíntesis , Proliferación Celular/genética , Ciclina B/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.
Asunto(s)
Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Análisis Mutacional de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Isocitrato Deshidrogenasa/genética , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Mutación , Nitrilos , Proteínas Nucleares/genética , Complejo Represivo Polycomb 2/genética , Mielofibrosis Primaria/mortalidad , Pronóstico , Pirimidinas , Proteínas Represoras/genética , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina , Resultado del TratamientoRESUMEN
Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Mutación , Trastornos Mieloproliferativos/genética , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/epidemiología , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Policitemia Vera/genética , Policitemia Vera/mortalidad , Policitemia Vera/patología , Mielofibrosis Primaria/epidemiología , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/mortalidad , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , Trombocitemia Esencial/genética , Trombocitemia Esencial/mortalidad , Trombocitemia Esencial/patologíaAsunto(s)
Proteínas de Unión al ADN/genética , Leucemia/genética , Mutación , Proteínas de Fusión Oncogénica/sangre , Factores de Transcripción/genética , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Complejo Represivo Polycomb 2 , Adulto JovenRESUMEN
To assess the clinical utility of targeted Next-Generation Sequencing (NGS) for the diagnosis of Inherited Retinal Dystrophies (IRDs), a total of 109 subjects were enrolled in the study, including 88 IRD affected probands and 21 healthy relatives. Clinical diagnoses included Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Stargardt Disease (STGD), Best Macular Dystrophy (BMD), Usher Syndrome (USH), and other IRDs with undefined clinical diagnosis. Participants underwent a complete ophthalmologic examination followed by genetic counseling. A custom AmpliSeq™ panel of 72 IRD-related genes was designed for the analysis and tested using Ion semiconductor Next-Generation Sequencing (NGS). Potential disease-causing mutations were identified in 59.1% of probands, comprising mutations in 16 genes. The highest diagnostic yields were achieved for BMD, LCA, USH, and STGD patients, whereas RP confirmed its high genetic heterogeneity. Causative mutations were identified in 17.6% of probands with undefined diagnosis. Revision of the initial diagnosis was performed for 9.6% of genetically diagnosed patients. This study demonstrates that NGS represents a comprehensive cost-effective approach for IRDs molecular diagnosis. The identification of the genetic alterations underlying the phenotype enabled the clinicians to achieve a more accurate diagnosis. The results emphasize the importance of molecular diagnosis coupled with clinic information to unravel the extensive phenotypic heterogeneity of these diseases.
Asunto(s)
Distrofias Retinianas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Proteínas del Ojo/genética , Femenino , Asesoramiento Genético/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Patología Molecular/métodos , Linaje , Retinitis Pigmentosa/genética , Adulto JovenRESUMEN
Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.