Association Between Gene Polymorphism Of BCL11A And Fetal Hemoglobin In β-Thalassemia Major In Guangxi Province

Objective To investigate the association between BCL11A gene polymorphism at rsl1886868, rs1427407, rs766432and HbF level and gamma-globin expression in hemoglobin β-thalassemia major patients in Guangxi province.Methods The peripheral blood of the unrelated β-thalassemia major subjects which divided into treatment group and untreated group and the normal controls in Guangxi were recruited and genomic DNA was extracted from2ml peripheral blood. BCL11A gene polymorphisms analysis at rs11886868, rs1427407and rs766432were detected by polymerase chain reaction(PCR), the PCR products were detected by restriction fragment length polymorphism(RFLP) and sequencing. Through counting genotypic frequency to learn about the differences between all patients and healthy controls, and analyze the association between the loci above and HbF level in the β-thalassemia major patients of untreated group. Total RNA in the single nuclear cells were extracted from2ml peripheral blood and were reversed transcription. The expression of different genotypes diversity of gamma-globin gene at rs766432of BCL11A gene in two groups was detected by utilizing SYBR Green I real-time fluorescence quantitative RT-PCR relative quantitative analysis method, which β-actin gene was employed as reference genes. The relative expression of ymRNA in different genotypes was analyzed by double-standard curves method of relative quantification PCR to analyze the association between different genotypes at this loci and gamma-globin expression.Results The genotypic distribution at rs1427407and rs766432in β-thalassemia major patients of untreated group were obviously displayed (P>0.001, P<0.001), the mutation frequency at these loci in β-thalassemia major subject were obviously higher than controls. While, there was no significant differences at rs11886868(P>0.05). There was significant correlation between genotypic distribution and three polymorphism loci in β-thalassemia major patients of two groups(P<0.001,Cp=0.477). There was no statistically significant in HbF level among different genotypes at rs11886868loci(P>0.05). Compared with the non-mutated genotype, the level of HbF in mutated genotype at rs1427407and rs766432in (3-thalassemia major patients of untreated group were higher, and there was significant differences in each genotypes (P<0.001, P<0.001). The correlation analysis showed that the gene polymorphisms were associated with HbF level(rp=0.931, P<0.001; rp=0.915, P<0.001). Moreover, we found, genetic mutations at rs766432was accompanied by mutations at rs1427407in the β-thalassemia major subjects and the normal controls, and most of the mutations type are no difference. Through analyzing different genotypes at rs766432in BCL11A gene of β-thalassemia major patients in treatment group and untreated group via fluorescent quantitative RT-PCR, there were significant differences in the relative expression of ymRNA in three genotypes in the two groups (P<0.001, P<0.05), and the relative expression of ymRNA in three genotypes in untreated group was diverse from each other. However, in the treatment group,the relative expression of ymRNA in CC was higher than the expression in AA. The correlation analysis showed that the gene polymorphisms were associated with the expression of Y mRNA (rp=0.924, P<0.001; rp=0.567, P<0.05)Conclusion (1) The mutation frequency at rs1427407and rs766432in BCL11A gene in β-thalassemia major subject of untreated group in Guangxi is obviously higher than controls. HbF level of mutated genotype at rs1427407and rs766432are higher than non-mutated genotype, which demonstrates that gene polymorphisms at two loci are related to HbF level, and gene mutations at rs1427407and rs766432may increase HbF level.(2) Genotypic distribution in (3-thalassemia major subject of treatment group and untreated group were significantly correlated with three polymorphism loci.(3) There may be individual overlaps between gene mutations at rsl427407and rs766432, and there may be some relevance between two loci gene mutations.(4) Gene polymorphisms at rs766432in (3-thalassemia major subject of treatment group and untreated group are related to the expression of Y mRNA, gene mutations could promoted gamma-globin expression,and the relation is significant in the untreated group.