RESUMEN
The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.
Asunto(s)
Epigénesis Genética , Perfilación de la Expresión Génica , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Talasemia beta/genética , gamma-Globinas/genética , Animales , Antígenos CD34/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Línea Celular , Niño , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Femenino , Hemoglobina Fetal/genética , Edición Génica , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Sulfitos , Secuenciación Completa del Genoma , Talasemia beta/patologíaRESUMEN
Calcium-activated chloride channels (CaCCs) play a vital role in regulating pulmonary artery tone during pulmonary arterial hypertension (PAH) induced by high blood flow. The role of CaCCs inhibitor niflumic acid (NFA) in vivo during this process requires further investigation. We established the PAH model by abdominal shunt surgery and treated with NFA in vivo. Fifty rats were randomly divided into normal, sham, shunt, NFA group 1 (0.2 mg/kg), and NFA group 2 (0.4 mg/kg). Pathological changes, right ventricle hypertrophy index, arterial wall area/vessel area, and arterial wall thickness/vessel external diameter were analyzed. Then contraction reactions of pulmonary arteries were measured. Finally, the electrophysiological characteristics of pulmonary arterial smooth muscle cells were investigated using patch-clamp technology. After 11 weeks of shunting, PAH developed, accompanied with increased right ventricle hypertrophy index, arterial wall area/vessel area, and arterial wall thickness/vessel external diameter. In the NFA treatment groups, the pressure and pathological changes were alleviated. The pulmonary artery tone in the shunt group increased, whereas it decreased after NFA treatment. The current density of CaCC was higher in the shunt group, and it was decreased in the NFA treatment groups. In conclusion, NFA attenuated pulmonary artery tone and structural remodeling in PAH induced by high pulmonary blood flow in vivo. CaCCs were involved and the augmented current density was alleviated by NFA treatment.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Ácido Niflúmico/uso terapéutico , Arteria Pulmonar/efectos de los fármacos , Circulación Pulmonar/fisiología , Remodelación Vascular/efectos de los fármacos , Animales , Arteriolas/efectos de los fármacos , Arteriolas/patología , Canales de Cloruro/antagonistas & inhibidores , Modelos Animales de Enfermedad , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Ácido Niflúmico/administración & dosificación , Ácido Niflúmico/farmacología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Ratas Sprague-Dawley , Túnica Media/efectos de los fármacos , Túnica Media/patologíaRESUMEN
INTRODUCTION: ß-thalassemia is a severe hereditary hemolytic anemia. Due to the diversity of mutations spectrum, ß-thalassemia manifests a highly heterogeneous clinical severity. We noted that a previous report characterized HBB:c.313delA, at the end of exon 2, as a ß-thalassemia trait rather than dominant ß-thalassemia, the classification given to similar mutations. We further explored the impact of this functional variant on globin structure through larger pedigree analysis and in vitro studies. METHODS: Hematological analysis and molecular genotyping were conducted on the proband and his family members. We evaluated functional effects of the variant on ß-globin gene in the proband's nucleated erythrocytes and transfected HEK-293T cells. Three-dimensional construction of protein structure was carried out in silico to demonstrate amino acid changes. RESULTS: The thalassemia major proband was identified as a compound heterozygote of HBB:c.313delA and HBB:c.126_129delCTTT. Three family members with heterozygotes of HBB:c.313delA displayed microcytic hypochromic anemia. Molecular characterization demonstrated that the frameshift mutation could give rise to retro-positioning of the termination codon, resulting in an elongated ß-globin chain with an extension of 10 amino acids. Clinical phenotype and functional experiments indicated that HBB:c.313delA led to ß0 -thalassemia phenotype. CONCLUSION: We concluded that the phenotype of HBB:c.313delA was mainly related to the stability of mutant mRNA, the degradation of mutant proteins, and production of inclusion bodies according to a systematic description of clinical phenotype and a series of molecular experiments.
Asunto(s)
Alelos , Fenotipo , Eliminación de Secuencia , Globinas beta/genética , Talasemia beta/sangre , Talasemia beta/genética , Sustitución de Aminoácidos , Índices de Eritrocitos , Eritrocitos/metabolismo , Eritrocitos/patología , Exones , Mutación del Sistema de Lectura , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Genotipo , Heterocigoto , Humanos , Cuerpos de Inclusión , Talasemia beta/diagnósticoRESUMEN
The correlation between calcium-activated chloride channel (CaCC) and pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow remains uncertain. In this study, we investigated the possible role and effects of CaCC in this disease. Sixty rats were randomly assigned to normal, sham, and shunt groups. Rats in the shunt group underwent abdominal aorta and inferior vena cava shunt surgery. The pulmonary artery pressure was measured by catheterization. Pathological changes, right ventricle hypertrophy index (RVHI), arterial wall area/vessel area (W/V), and arterial wall thickness/vessel external diameter (T/D) were analyzed by optical microscopy. Electrophysiological characteristics of pulmonary arterial smooth muscle cells (PASMCs) were investigated using patch clamp technology. After 11 weeks of shunting, PAH and pulmonary vascular structural remodeling (PVSR) developed, accompanied by increased pulmonary pressure and pathological interstitial pulmonary changes. Compared with normal and sham groups, pulmonary artery pressure, RVHI, W/V, and T/D of the shunt group rats increased significantly. Electrophysiological results showed primary CaCC characteristics. Compared with normal and sham groups, membrane capacitance and current density of PASMCs in the shunt group increased significantly, which were subsequently attenuated following chloride channel blocker niflumic acid (NFA) treatment. To conclude, CaCC contributed to PAH induced by high pulmonary blood flow and may represent a potential target for treatment of PAH.