Effects Of Modifier Genes On Hematological Phenotypes In Thalassemia

Objective This study is based on the samples of thalassemia in Fujian Province and relevant test data.A series of representative thalassemia modifier genes was selected for population screening to compare these genetic locus and their effects on hematological phenotype.The function of some genetic locus is discussed in order to provide new ideas for the diagnosis and treatment of thalassemia.Methods 1.Multiplex PCR combined agarose gel electrophoresis and PCR-RBD were used to detect thalassemia ? and ? genes in 463 individuals with alpha and beta thalassemia in Fujian Province;blood and hemoglobin electrophoresis,iron metabolism and other relative tests were performed on the samples in order to collect the parameters of each hematological phenotype.According to the thalassemia genotype and Hb F > 1.5%,the samples were divided into 7 groups of A-G.Sanger sequencing was used to detect the KLF1 gene in each sample collected.2.Screening by Haplo View 4.2 and using high-resolution melting curve(HRM)method to detect the tag polymorphic loci on the HBG1,HBG2,HBS1L-MYB,and BCL11 A genes,and comprehensively analyze the correlation between polymorphic loci and phenotypic parameters of thalassemia patients.3.Replace the promoter region of the reporter gene plasmid(p GL3-basic)with a specific fragment of the target promoter to construct a mutant and normal recombinant plasmid,and transfect the constructed plasmid into the target cell for promoter luciferase assay.Results 1.A total of 371 patients or carriers of alpha and beta-thalassemia genes were detected.The analysis found that the genotype distribution of thalassemia in Fujian Province has its own characteristics.rs3817621,rs 2072597,rs 2072596,rs117351327,c.519-525 dup CGGCGCC,c.895C> G,c.472G> A and c.1001C> G and other KLF1 gene mutation locus were detected.Among them,there was a linkage disequilibrium between rs3817621 and rs 2072597 and rs117351327.Some of the intermediate-thalassemia patients with KLF1 variants had Hb F levels that were different from wild-type samples.Hb A2,MCV,MCH and other hematological parameters have not yet seen differences.2.The rs4895441 in the HBS1L-MYB gene and rs3817621 in the KLF1 gene of ?-thalassemia patients are all related to the level of Hb F.The rs368698783 in the HBG1 gene,rs7482144 in the HBG2 gene,rs28384513 in the HBS1L-MYB gene,and rs4671393 in the BCL11 A gene of patients with ?-thalassemia were not detected and correlated with Hb F levels.There is linkage disequilibrium between rs368698783 in HBG1 gene and rs7482144 in HBG2 gene;the unbalanced linkage between rs28384513 and rs4895441 in HBS1L-MYB gene is weak.3.The transfection was in good condition and the dual luciferase reporter gene system was successfully constructed.Compared with the wild type control group,the fluorescence intensity of the c.-251 C > G mutation group was decreased,and the luciferase activity was decreased.Conclusions 1.KLF1 variation has an impact on the hematological phenotype of patients with various degrees of thalassaemia.The genetic variation is different from other regions.2.Modification of polymorphic loci can affect changes in Hb F levels in hematological phenotypes in patients with thalassemia.In the diagnosis of thalassemia,screening for polymorphic loci on the modified gene may help identify difficult cases.3.c.-251 C > G is associated with a decrease in KLF1 promoter activity,and its corresponding SNP should not be considered as a neutral mutation.