RESUMEN
Prenatal screening of ß-thalassemia (ß-thal) carriers is based on the hallmark phenotype of microcytosis and raised Hb A2. The unanticipated birth of ß-thal major (ß-TM) offspring to ß-thal carriers who were misdiagnosed during prenatal screening have been reported. A subset of these resulted from the masked phenotype due to the coinheritance of HBD variants. In a broader sense, the causes of reduced Hb A2 in thalassemia screening, the prevalence and spectrum of HBD variants in Hong Kong remain to be characterized. Over a 13-month period, a total of 2982 samples were referred for thalassemia screening. Surplus samples with reduced Hb A2 levels (2.0%) were evaluated. HBD variations were assessed by direct sequencing. Sixty-six samples were tested. Hb H disease, HBD variants, α-thalassemia (α-thal) trait and iron deficiency were detected in 40 (60.6%), 12 (18.2%), eight (12.1%) and seven (10.6%) samples, respectively. Seven samples carried more than one of the mentioned conditions. The cause remained elusive in seven samples. Thirteen HBD variants were detected and two were recurrent, including HBD: c.-127T>C [-77 (T>C)] and HBD: c.314G>A (Hb Chori-Burnaby). A novel nonsense variant HBD: c.262C>T [codon 87 (C>T)] was detected in cis with HBD: c.-127T>C. Overall, the prevalence of HBD variants was 0.4%. This study advanced our understanding of the causes of reduced Hb A2 in clinical practice and identified hereditary disorders of α- and δ-globin genes as the prevailing causes. It established the landscape of HBD variations in our locality and highlighted the pitfall of phenotypic screening of ß-thal carriers.
Asunto(s)
Talasemia alfa , Talasemia beta , Globinas delta , Hemoglobina A2/genética , Heterocigoto , Hong Kong/epidemiología , Humanos , Mutación , Talasemia alfa/diagnóstico , Talasemia alfa/epidemiología , Talasemia alfa/genética , Talasemia beta/diagnóstico , Talasemia beta/epidemiología , Talasemia beta/genética , Globinas delta/genéticaRESUMEN
RUNX1 encodes a Runt-related transcription factor that is critical for hematopoiesis. In this study, through a combinatorial molecular approach, we characterized a novel t(5;21)(q13;q22) translocation involving RUNX1 that was acquired during the progression of myelodysplastic syndrome to acute myeloid leukemia (AML) in a pediatric patient. We found that this translocation did not generate RUNX1 fusion but aberrantly upregulated RUNX1. This upregulation was attributed to the disruption of long-range chromatin interactions between the RUNX1 P2 promoter and a silencer in the first intron of the gene. Characterization of the silencer revealed a role of SNAG repressors and their corepressor LSD1/KDM1A in mediating the effect. Our findings suggest that chromosomal rearrangements may activate RUNX1 by perturbing its transcriptional control to contribute to AML pathogenesis, in keeping with an emerging oncogenic role of RUNX1 in leukemia.
Asunto(s)
Cromosomas Humanos Par 11/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Regulación hacia Arriba , Preescolar , Cromosomas Humanos Par 21/genética , Regulación Leucémica de la Expresión Génica , Humanos , Masculino , Regiones Promotoras Genéticas , Translocación GenéticaRESUMEN
Helicase-like transcription factor is a SWI/SNF chromatin remodeling factor involved in various biological processes. However, little is known about its role in hematopoiesis. In this study, we measured helicase-like transcription factor mRNA expression in the bone marrow of 204 adult patients with de novo acute myeloid leukemia. Patients were dichotomized into low and high expression groups at the median level for clinicopathological correlations. Helicase-like transcription factor levels were dramatically reduced in the low expression patient group compared to those in the normal controls (n=40) (P<0.0001). Low helicase-like transcription factor expression correlated positively with French-American-British M4/M5 subtypes (P<0.0001) and complex cytogenetic abnormalities (P=0.02 for ≥3 abnormalities;P=0.004 for ≥5 abnormalities) but negatively with CEBPA double mutations (P=0.012). Also, low expression correlated with poorer overall (P=0.005) and event-free (P=0.006) survival in the intermediate-risk cytogenetic subgroup. Consistent with the more aggressive disease associated with low expression, helicase-like transcription factor knockdown in leukemic cells promoted proliferation and chromosomal instability that was accompanied by downregulation of mitotic regulators and impaired DNA damage response. The significance of helicase-like transcription factor in genome maintenance was further indicated by its markedly elevated expression in normal human CD34(+)hematopoietic stem cells. We further demonstrated that helicase-like transcription factor was a RUNX1 target and transcriptionally repressed by RUNX1-ETO and site-specific DNA methylation through a duplicated RUNX1 binding site in its promoter. Taken together, our findings provide new mechanistic insights on genomic instability linked to helicase-like transcription factor deregulation, and strongly suggest a tumor suppressor function of the SWI/SNF protein in acute myeloid leukemia.
Asunto(s)
Células de la Médula Ósea/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/genética , Antígenos CD34/metabolismo , Sitios de Unión , Células de la Médula Ósea/patología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Aberraciones Cromosómicas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Femenino , Inestabilidad Genómica , Hematopoyesis/genética , Humanos , Cariotipo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Mutación , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoAsunto(s)
Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 3/genética , Fibronectinas/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Fusión Oncogénica/genética , Receptor alfa de Ácido Retinoico/genética , Translocación Genética , Adulto , Cromosomas Humanos Par 17/metabolismo , Cromosomas Humanos Par 3/metabolismo , Fibronectinas/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Masculino , Proteínas de Fusión Oncogénica/metabolismo , Receptor alfa de Ácido Retinoico/metabolismoRESUMEN
The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.