RESUMO
Increased levels of fetal hemoglobin (HbF: α2γ2) can ameliorate the clinical severity of the ß-hemoglobinopathies. Microarray analysis represents a powerful approach to identify novel genetic factors regulating the γ-globin gene. Gene expression profiling was previously performed on 14 individuals with high or normal HbF levels to identify the genetic factors that control γ-globin gene expression. To obtain more accurate and reliable results, our results were combined with public microarray dataset GSE22109 deposited in the Gene Expression Omnibus database. Annotation of case versus control samples was taken directly from the microarray documentation. The differentially expressed genes (DEGs) were obtained and were deeply analyzed by bioinformatics methods. Combined with our own chip expression data, potential genes HBE1, TFRC, and CSF2 were selected out for subsequent qRT-PCR validation. A total of 184 DEGs were identified from GSE22109 and the protein-protein interaction network was constructed. Gene set enrichment analysis showed that the hematopoietic cell lineage pathway overlaps in the two datasets. HBE1, CSF2, and TFRC were confirmed by qRT-PCR. Our results suggest novel candidate genes and pathways associated with the γ-globin gene expression.
Assuntos
Antígenos CD/genética , Biomarcadores/sangue , Hemoglobina Fetal/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Hemoglobinopatias/patologia , Receptores da Transferrina/genética , Globinas beta/genética , Adulto , Estudos de Casos e Controles , Biologia Computacional , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hemoglobinopatias/sangue , Hemoglobinopatias/epidemiologia , Hemoglobinopatias/genética , Humanos , Masculino , Análise em Microsséries , Prognóstico , Mapas de Interação de ProteínasRESUMO
Foetal haemoglobin (HbF) plays a dominant role in ameliorating the morbidity and mortality of ß-thalassaemia. A better understanding of the loci and genes involved in HbF expression would be beneficial for the treatment of ß-thalassaemia major. However, the genes associated with HbF expression remain largely unknown. In this study, we first explored large-scale data sets and examined the human genome for evidence of positive natural selection to screen out single nucleotide polymorphisms (SNPs). A genetic analysis of HbF levels was conducted in a Chinese cohort of patients with ß-thalassaemia to confirm the bioinformatics results. A total of 1141 subjects with ß-thalassaemia were recruited. The results showed that the SNP rs11759328 in the ARHGAP18 gene was significantly associated with HbF levels (Ρ = 5.1 × 10-4). ARHGAP18 belongs to the RhoGAP family and controls angiogenesis, cellular morphology and motility. Second, after determining that ARHGAP18 was highly expressed in the human K562 cell line, we used lentiviral-mediated small interfering RNA to knock down ARHGAP18 expression and subsequently assessed cell proliferation and apoptosis using cell proliferation assays and flow cytometry, respectively. ARHGAP18 downregulation in K562 cells significantly increased HBG1/2 expression and apoptosis, but proliferation was not significantly affected in vitro. Our data suggest that ARHGAP18, which was located by the SNP rs11759328 via positive selection, plays a potential role in regulating HbF expression in ß-thalassaemia and may be a promising therapeutic target. Knockout studies of ARHGAP18 warrant further investigation into its aetiology in HbF.
Assuntos
Hemoglobina Fetal/genética , Proteínas Ativadoras de GTPase/genética , Seleção Genética/genética , Talassemia beta/genética , Adolescente , Adulto , Apoptose/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Estudos de Associação Genética , Humanos , Células K562 , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto Jovem , Talassemia beta/sangue , Talassemia beta/patologiaRESUMO
ß-Thalassemia (ß-thal) is the most common inherited hemolytic anemia worldwide. Elevated Hb A2 is a mark of ß-thal carriers. The aim of this study was to identify the pathogenic variants associated with the Hb A2 levels. One thousand and thirty ß-thal carriers were recruited for this study. Using positive natural expression quantitative trait loci (eQTL) analysis, a significant variant was selected. Genotyping for the rs231841 polymorphism was performed by the Sequenom MassARRAY IPLEX platform. All genetic association analyses were performed with the PLINK program. The linear regression analysis showed that rs231841 in the intron region of the potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene on chromosome 11p15 was significantly associated with Hb A2 levels. The presence of the C allele was associated with elevated Hb A2 levels. Our results suggest that rs231841 on the KCNQ1 gene with positive natural selection is related to Hb A2 levels in Chinese ß-thal carriers, and KCNQ1 is probably associated with the expression of the ß-like globin gene cluster.
Assuntos
Povo Asiático/genética , Variação Genética , Hemoglobina A2/genética , Seleção Genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adolescente , Adulto , Idoso , Alelos , Criança , Pré-Escolar , China , Índices de Eritrócitos , Heterozigoto , Humanos , Pessoa de Meia-Idade , Família Multigênica , Mutação , Polimorfismo de Nucleotídeo Único , Adulto Jovem , Globinas beta/genéticaRESUMO
The implications of lncRNAs regarding fetal hemoglobin (HbF) induction in hemoglobin disorders remain poorly understood. In this study, microarray analysis was performed to profile lncRNAs, miRNAs and mRNAs in individuals with hereditary persistence of fetal hemoglobin (HPFH), ß-thalassemia carriers with high HbF levels and healthy controls. The results show aberrant expression of 862 lncRNAs, 568 mRNAs and 63 miRNAs in the high-HbF group compared with the control group. Altered NR_001589, NR_120526, T315543, miR-486-3p, miR-19b-1-5p and miR-20a-3p expression was confirmed by quantitative reverse transcription-polymerase chain reaction, and Spearman correlation coefficients revealed significant positive correlations with HbF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed the hematopoietic cell lineage and apoptosis to be most significantly dysregulated in HbF induction. We analyzed coding genes near the lncRNAs and constructed a coding-noncoding co-expression network. Based on the results, lncRNAs likely contribute to increased HbF levels by activating expression of HBE1 and hematopoietic cell lineage-inducible molecules and by inhibiting that of apoptosis-inducible molecules. Finally, through construction of a competing endogenous RNA network, we found that 6 lncRNAs could bind competitively with miR-486-3p, resulting in increased HbF levels. Taken together, our findings provide new insights into the mechanisms of HbF induction and potentially provide new targets for the treatment of ß-thalassemia major.