Association GT Gene Promoter-158C/T Polymorphism with Methylation Status in B-thalassemia Major
|School||Guangxi Medical University|
|Keywords||β-thalassemia major γ-hemoglobin gene mRNA expression ~Gγ-158C/T polymorphism DNAmethylation|
Objective:This study was aimed to examine Gγ-158C/T polymorphism and the promoter CpG island methylation of γ-globin gene in peripheral blood mononuclear cells fromβ-thalassemia major and health adult in Guangxi province,then to investigate the rate of the promoter methylation in these two objects of study. Meantime, to detect γ-hemoglobin gene mRNA expression. Eventually,to identify preliminarily γ-globin gene promoter methylation sites which may be relevant to the expression of γ-globin. To discuss the methylation state,γ-hemoglobin gene mRNA expression and Gγ-158C/T polymorphism of relevancy.Methods:Collected4ml blood of β-thalassemia major patients and healthy, then extracted DNA and RNA from peripheral blood mononuclear cells. Bisulfite genomic sequencing PCR (BSP) technique was used to detect quantitatively methylation of the promoter CpG sites in γ-globin gene,through bisulfite-modification directed Touchdown PCR(TD-PCR) products to DNA clone sequencing. Specimen RNA into cDNA transcriptase used in real-time quantitative fluorescence PCR (SYBR GREEN relative quantitative) test γ-hemoglobin gene mRNA expression.Results:1.87cases of y-158C/T polymorphism analysis:β-thalassemia major patients Gγ-158sites heterozygous mutation rate of significantly higher than normal11.49%vs1.15%(X2=7.861, P=0.009, P<0.05), a statistically significant difference.2. The results indicated that hypermethy lation of γ-globin gene promoter CpG island both occurred in β-thalassemia major and health adult. The DNA methylation status of4CG sites within the γ-globin promoter region was analyzed, in the series which located in28bp,122bp,231bp,234bp.3.30cases of β-thalassemia major specimen methylation states:β-thalassemia major patients, and normal γ-hemoglobin gene promoter methylation sites each area CG are in highly methylation level, of which122bp site methylation rate was significantly lower than the normal control group (X2=10.784, P<0.01);231bp site methylation rates lower than normal control group (X2=4.110, P<0.05). There are both no significant difference in28bp and234bp sites(P>0.05).4.10cases of merger XmnI+/-β-thalassemia major specimens γ protein gene expression mRNA detection:Merger XmnI+/-β-thalassemia major specimens and normal control group γ-hemoglobin gene expression significant differences (Gγ:Z=-3.780,P=0.000;Aγ:Z=-2.851,P=0.004) are statistically significant differences.According to the mRNA=2-ΔΔct, with the median calculation Gγ mRNA about normal controls expression of44times, Aγ of about10times as much as normal.5.10cases of merger XmnI+/-β-thalassemia major specimens to the correlation analysis:158polymorphisms and express related analysis results of mRNA Gγ hemoglobin gene (γ=0.867, P=0.000), Aγ hemoglobin gene (γ=0.451, P=0.046) mRNA expression and XmnI+/-positive correlation;Gγ hemoglobin gene mRNA expression and γ gene promoter methylation rates are negative correlation (γ=-0.777,P=0.008, P<0.01), Aγ mRNA expression and γ gene promoter methylation rates unrelated (γ=-0.068,P=0.851, P>0.05).Conclusion: B-thalassemia major-158sites heterozygous mutation rate of significantly higher than normal;The methylation sites28bp,122bp,231bp and234bp of γ-globin gene promoter can be found both in β-thalassemia major and health adult.Carry-158mutations β-thalassemia major, γ-hemoglobin gene mRNA expression and Gγ revealed in Aγ increase; B-thalassemia major patients γ gene promoter methylation relatively normal state area is low, Gγ mRNA expression negatively related with y gene promoter methylation rates.And then the preliminary identification of122bp and231bp sites participate in the regulation of γ-globin expression would provide a basis for further demethylation which may be the new target for gene therapy in β-thalassemia major.