| THE FIRST PART OF THESIS Correlation between phenotype and genotype of non-transfusion dependentβ-thalassemia patientsObject: 1.The aim of this study was to investigate the relationship between the clinical phenotype and genotype non-transfusion dependent beta-thalassemia patients(TI phenotypes)by statistically analyzed the data of clinical features and molecular basis.2.To explore the relationship between the polymorphisms of HBG2,BCL11 A,HBS1L-MYB genes and the level of hemoglobin F(Hb F);3.To explore the relationship among the mRNA expression of AHSP,KLF1,KLF2,BCL11 A and globin genes and the level of hemoglobin.Methods:A total of 102 patients with TI phenotypes(57 males,45females)were recruited for this study from June 2013 to February 2017 from the first affiliated hospital of Guangxi Medical University,and 60 healthy controls(33 males and 27females)from physical examination center of our hospital were chosen.The median age of the TI patients was 22(2-57)years old,and the median age of the controls was 21(4-55)years old.The clinical data and blood sample were collected,and complete blood count(CBC),biochemical analysis and hemoglobin electrophoresis was conducted on the all TI patients;the α-and β-globin gene was determined using Polymerase Chain Reaction(PCR)sequencing,gap PCR,real time PCR and Next Generation Sequencing(NGS);serum ferritin was measured by electrochemiluminescence(ELCA);the liver R2 and cardiac T2* was detected by magnetic resonance imaging(MRI),and analyzed by Ferriscan? and CMR software.The polymorphisms of HBG2,BCL11 A and HBS1L-MYB genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and DNA sequencing;the real-time PCR was used to detect the mRNA expression ofα-,β-,γ-globin gene and AHSP,KLF1,KLF2,BCL11 A gene;and their clinical data,test results were analyzed and compared.Result:1.In the 102 TI patients 77(75.5%)patients had transfusion treatment,and the median volume of blood transfusion was 800ml(range 0-20000ml),the mean Hb was 76.47±17.13g/L,the median Hb F% was 36.9%(range1.1-97.6%).36(35.3%)patients underwent splenectomy,and 28(27.7%)patients received iron chelation treatment.The median level of SF was1481.5 ng/m L(range 27-8300 ng/m L),LIC was 14.4 mg/g.dw(rage 2-43mg/g.dw),among them 94.5% patients was iron overload,the media levels of cardiac T2* was 35.64 ms(15.53-75.1 ms),only 4.6% patients was cardiac iron overload.2.The frequency and spectrum of β-thalassemia mutations of 98 TI phenotypes patients was different,the most common mutations was 41-42(-TTCT),-28(A->G),CD 26(G->A),17(A->T).83(84.7%)patients was homozygous or compound heterozygous β-thalassemia alleles(A genetype),including19(22.9%)homozygous,33(39.7%)associated with Hb E and 19(22.9%)with α-thalassemia,and 15(15.3%)was heterozygosity(B genetype),including 1 patient with α-thalassemia mutation,3 patients withα-thalassemia copy contain 1 patient associated with Hb Broomhill,the difference of hematology parameters(Hb、MCV、MCH、Hb F and Hb A2)and iron metabolism parameters was analyzed,there were significant difference on the Hb F(40 VS 4.2,P<0.001),MCH(22VS25.79,p=0.003)and AST(37vs25.5,p=0.039)between the two groups;and there were no significant difference on the Hb and iron metabolism parameters.The difference of the hematology parameters of the two groups patients with or without α-thalassemia were not significant.3.There was no correlation between SNP locus genotype and Hb A2,Hb F and Hb levels and clinical severity in patients with non-transfusion-dependentβ-thalassemia.Multiple linear regression analysis showed that Hb F and rs766432 C→A mutation were influenced Hb level.Multiple log regression analysis showed that the possibility of severe clinical phenotype of β0 / β0genotype was increased 6.39 times than β+/β+,and β+/ β0 genotype increased 5.13 times than β+/β+.4.There were significantly increased of the mRNA expression of AHSP and BCL11 A genes and the ratio of α/β(p<0.05);but the difference of KLF1 and KLF2 were not significant(p>0.05).There were no difference on the mRNA expression of AHSP,KLF1,KLF2 and BCL11 A gene among the different clinical severity of TI thalassemia and among the A and B genetype(p>0.05).AHSP and KLF1 was positively correlated withγglobin gene(r=0.77,p<0.01;r=0.41,p<0.01),negatively correlated with Hb(r=-0.293,p=0.01;r=-0.34,p=0.004);KLF1 was negatively correlated with Hb and Hb F(r=-0.28,p=0.018;r=-0.27,p=0.027);BCL11A was positively correlated with KLF1 and γglobin gene(r=0.28,p=0.028).Conclusions1.TI phenotype patients in southern China was phenotypic diversity and genotypic heterogeneity.2.β0-thalassemia mutations was a major factor affecting the clinical phenotype of non-transfusion-dependent β-thalassemia.3.Among the 98 TI phenotype patients,there are still some patients failed to clarify their molecular basis,which need further study.THE SECOND PART OF THESIS Effect of decitabine on epigenetic modification at γ-globin gene promoter in K562 cellsObjective: To gain more insight into the mechanism of action and the effect of decitabine on epigenetic modification at γ-globin gene promoter in K562 cells.Methods: Three groups were set up in this study,including decitabine group,hydroxyurea(HU)positive group and dimethyl sulfoxide + PBS negative control group.Expression levels of globin gene mRNA were assessed by real-time PCR,and apoptosis rate were detected by flow cytometry,then selected the optimal concentration and time,and the selected experiment conditions were used in the following study.α-and β-globin protein level were assessment by Western blot analysis,enrichment of histone marks included dimethyl-histone H3(lys4)(H3K4me2),acetylated histone H3(ac H3),acetylated histone H4(ac H4)at γ-globin gene promoter was assessed by chromosome immunoprecipitation(Ch IP).DNA methylation levels on human gamma-globin gene promoters were determined using pyrophosphate sequence analysis.Results: 1.Screening of optimal concentration and times of Decitabine,the results showed that the difference of γ-globin gene mRNA expression at different time and concentration were significant(F=32.3,p<0.001,F=5.37,p=0.047),with no interaction between concentration and times for γ-globin gene,The difference of α-and β-globin gene mRNA expression at different time and concentration were not significant(F=2.02,P=0.23,F=0.58,p=0.639),with no interaction between concentration and times for α-and β--globin gene(F=21.72,p<0.001;F=13.86,p=0.001).The inductive effect of γ-globin gene of decitabine at 400 n M for 48 h is optimal for K562 cells.2.The apoptosis rate of decitabine at different times in K562 cells were significantly different(p<0.05),but not significant for the concentration,and without interaction between concentration and times for apoptosis rate(F=0.181,p=0.349).3.The difference of α-and γ-globin gene mRNA expression with decitabine and HU treatment were significant(p=0.037,p=0.02),and the difference of α-gene mRNA expression between control vs decitabine group and control vs HU were significant(p=0.018,p=0.037),but with no difference between decitabine and HU(p=0.347).The difference of γ-globin gene mRNA expression between groups were significant.There were no significantly difference of β-globin mRNA expression and ratio of globin gene with different treatment between groups(p>0.05).).4.Western blotting analysis showed that the difference of the protein levels ofγ-globin with different treatment were significant(p<0.001),compare with control,decitabine treatment increased 2.84 fold,and HU treatment increased1.15 fold.5.The levels of H3K4me2 of γ-globin gene promoter were lower in K562 cells with different treatment compared with control but no significant(p=0.15),the levels of ac H3 and ac H4 among the three groups were significant(p<0.001,p=0.012),the difference of the levels of ac H3 between control vs HU and decitabine vs HU were significant(p=0.007,p=0.009),but no significant between decitabine and HU(P=0.116).For the levels of ac H3,there was no significant difference between decitabine and HU(P=0.073).6.The average degree of DNA methylation of 6 Cp Gs of γ-globin gene promoter with different treatment were lower than control,but not significant(p>0.05).7.Both the levels of γ-globin gene mRNA expression and acetylation increased with decitabine and HU treatment compare to the control,but the the levels of H3K4me2 and DNA methylation decreased.Conclussion: 1.Decitabine and HU increased the α-and γ-globin gene mRNA and γ-globin protein expression in K562 cells.2.Decitabine and HU significantly increased γ-globin gene promoter ac H4 levels in K562 cells,but the increase of ac H3 levels was not significant.3.The levels of H3K4me2 and DNA methylation were not significantly decreased with dicetabine and HU treatment.THE THIRD PART OF THESIS Effect of decitabine on epigenetic modification at γ-globin gene promoter in humanized non-transfusion dependent thalassemia mouse modelObjective: The aim of the study was to gain more insight into the mechanism of action of decitabine and the effect of the drug on epigenetic modification atγ-globin gene promoter in humanized non-transfusion dependent thalassemia mouse model.Methods: Generating humanized non-transfusion dependent thalassemia mouse model,and divided into decitabine group,HU positive group and normal saline mouse model group(Mork)three groups.Expression levels of globin gene mRNA were assessed by real-time PCR,apoptosis rate were detected by flow cytometry,enrichment of histone marks including H3K4me2,ac H3,ac H4 atγ-globin gene promoter was assessed by Ch IP.Results: 1.The levels of α-globin gene mRNA expression with different treatment lower than Mork(p=0.017),but the difference between dicitabine and HU were not significant?(p=0.346),β-and γ-globin mRNA expression significantly higher than mork(p<0.01,p<0.01),ratio α-to non-α-globin mRNA expression decreased compared to Mork,but there was no significant difference between decitabine and HU(p=0.435).2.The apoptosis rate of bone marrow cells with different treatment lower than Mork(p<0.001),but there was no significant difference between decitabine and HU(p=0.435).3.The levels of H3K4me2 at γ-globin gene promoter was lower than Mork,and there was no significant difference between decitabine and HU(p=0.771),the levels of ac H3,ac H4 was significantly increased in drug treatment than mork(p<0.001,p<0.001),and the difference between decitabine and HU were significant(p=0.008,p=0.006).4.Both the levels of γ-globin gene mRNA expression and acetylation increased with decitabine and HU treatment compare with the control,but the the levels of H3K4me2 decreased.Conclusion: 1.Decitabine and HU increased the β-and γ-globin gene mRNA expression,decreased the α-globin gene mRNA expression in humanized non-transfusion dependent thalassemia mouse model.2.Decitabine and HU decreased the apoptosis rate of bone marrow cells in humanized non-transfusion dependent thalassemia mouse model.3.Decitabine and HU increased γ-globin gene promoter ac H3 and ac H4 levels,decreased the levels of H3K4me2 in humanized non-transfusion dependent thalassemia mouse model. |
