DNA Polymorphisms At HBS1L-MYB, BCL11A And HBG Associate With Fetal Hemoglobin Quantitative Trait

PART 1 DNA POLYMORPHISMS AT HBS1L-MYB, BCL11A AND HBG ASSOCIATE WITH FETAL HEMOGLOBIN QUANTITATIVE TRAITObjectives To identify the genetic mechanics that influence fetal hemoglobin (HbF) and other hematologic parameters, we performed a replicated study following the genome-wide association study (GWAS) guidelines. We identified 4 independent regions of interest, HBS1L-MYB intergenic region, BCL11A locus, HBG2 gene cluster, and the CSNK2A1 gene and analysed their association with the level of HbF and other hematologic parameters.Methods 312 unrelatedβ-thalassemia subjects in Guangxi were recruited and phenotyped. Hemoglobin type and quantity, along with red blood cell (RBC) count and hemoglobin (Hb) were assessed. Single-nucleotide polymorphism (SNP) analysis was performed by using PCR/restriction enzymes and sequencing. Hardy–Weinberg (H-W) equilibrium and linkage disequilibrium (LD) patterns of the interested SNPs were analyzed using Haploview v.4.2. Association of SNPs with HbF level and other hematologic parameters was analysed using SPSS version 16.0. Haplo.state software was used to infer significant haplotypes.Results 10 SNPs were associated with HbF level. 7 SNPs in HBS1L-MYB gene (chr.6q23) were significantly associated with HbF level and 1 SNP was associated with RBC count and Hb. 2 SNPs in BCL11A gene (chr.2p16.1) were significantly related with HbF level. 1 SNPs in the upstream of HBG2 promoter region (chr.11) showed significant association with HbF. In addition, haplotypes of HBS1L-MYB and BCL11A were identified and showed association with HbF production. We also found a significant impact of HBS1L- MYB haplotypes on RBC count and Hb. Haplotypes of HBG2 and BCL11A were not impact on RBC count and Hb. rs6037828 in CSNK2A1 gene was not associated with hematologic parameters.Conclusion 3 independent regions, including HBS1L-MYB intergenic region, BCL11A locus, and HBG2 gene cluster, were associated with hematologic parameters. This study can significantly improve the GWAS findings in patients withβ-thalassemia of Guangxi province. and is useful for further research in the field of common predictors of the erythropoiesis. PART2 LEVELS OF BCL11A EXPRESSION ON PERIPHERAL BLOOD MONOCYTES OF PATIENTS WITHβ-THALASSEMIA MAJORObjectives To explore the levels of B-cell CLL/lymphoma 11A (BCL11A) mRNA relative expression on peripheral blood mononuclear cells(PBMCs) in patients withβ-thalassemia major. To find out there are features of BCL11A mRNA expression.Methods 35 patients withβ-thalassemia major were involved into case group. 40 healthy controls were involved into control group. Red blood cell (RBC) count and hemoglobin (Hb) were also tested. Total RNA samples from fresh PBMCs in the peripheral blood were reverse transcribed into cDNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to internal reference gene. SYBR Green I real-time fluorescence quantitative PCR (RT-PCR) and relative quantitative analysis method were performed to detect the mRNA expression of BCL11A in PBMCs of 2 groups. Relative expression of mRNA in 2 groups were compared by 2-△△Ct method of relative quantification. Bivariate correlation analysis between BCL11A gene mRNA relative expression with red RBC count, Hb, age or sex was used. Normal distribution of measurement data was statisticed by Pearson correlation analysis and the other data was statisticed by Spearman correlation analysis.Results The levels of BCL11A expression in control group were higher than case group. In the case group, the lowest expression level of BCL11A mRNA was 0.81 times as much as calibrator and the highest was 2.28 times higher than calibrator. In the control group, the lowest expression level of BCL11A mRNA was 2.29 times higher than calibrator and the highest was 4.92 times. Individual differences of relative expression in BCL11A mRNA were obvious. Relative expression of BCL11A gene mRNA did not associated with RBC count, Hb, sex or age.Conclusion The levels of BCL11A mRNA relative expression were decreased in patients withβ-thalassemia major. Low expression of BCL11A mRNA may be contributed to persistent expression ofγ-globin gene.