GDF-15 Gene Polymorphism And Comparative Serum Glycoprotein Of Iron Overload In Thalassemia

CHAPTER Ⅰ Association between GDF-15 Gene Polymorphism and Risk of Susceptibility to iron overload in patients of thalassemiaObjective To study the single nucleotide polymorphisms(SNP)of GDF-15 gene at position rsl058587,rsl059369 and rs4808793 in healthy population and iron overload in thalassemia patients.To investigate whether rsl058587,rsl059369 and rs4808793 polymorphisms are associated with susceptibility to iron overload in thalassemia patients.Methods The polymorphisms of rsl058587,rsl059369 and rs4808793 were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method(PCR-RFLP)in 122 patients with iron overload in thalassemia patients and 162 no-iron overload in thalassemia patients and128 healthy controls.The χ2 test was used to analyze whether the distribution of the SNP genotype frequencies in patients group and the healthy controls group consistent with Hardy-Weinberg equilibrium(HWE).Binary logstic regression was used to test the associations between polymorphisms and susceptibility to iron overloaded in thalassemia patients.Results The genotype distribution of polymorphism of rs1059369 and rs4808793 in both groups was consistent with the Hardy-Weinberg equilibrium,that means the samples were representative and comparable(P>0.05).However,the genotype distribution of polymorphism of rs1058587 showed P<0.001,which failures to meet the examination of Hardy-Weinberg equilibrium.For rs1059369,statistical analysis showed significantly differences in genotypic frequencies distribution between iron overloaded in thalassemia patients,no-iron overloaded in halassemia patients and healthy subjects(P<0.05).The Tallelic requency was higher in healthy controls compared with iron overloaded in thalassemia patients(P<0.05).Nevertheless,comparing no-iron overloaded in thalassemia patients with iron overloaded in thalassemia patients and healthy controls,statistical analyses showed no significant differences in both groups(P>0.05).Moreover,overloaded in thalassemia patients had significantly increased chance carrying A allele(OR=2.259,95%CI=1.177-3.031,P=0.003)also AA genotype and AT genotype(OR=2.297,95%CI=1.158-3.1014,P=0.002)compared with no-iron overloaded in thalassemia patients and healthy population.For rs4808793,statistical analysis showed significantly differences in genotypic frequencies distribution between iron overloaded in thalassemia patients,no-iron overloaded in thalassemia patients and healthy subjects(P<0.05)whereas it’s showed no differences in genotypic frequencies distribution between both of experimental groups(P>0.05).Nevertheless,overloaded in thalassemia patients had significantly increased chance carrying C allele(OR=1.295,95%CI=0.557-1.631,P=0.005)also CC genotype and CG genotype(OR=2.297,95%CI=1.158-3.1014,P=0.002)compared with no-iron overloaded in thalassemia patients and healthy population.Besides,for further analysis,dominance model and recessive model were performed,which showed significantly differences(P=0.013)in the person carrying C allele(CC+CG)increased in healthy controls,no-iron overloaded in thalassemia patients and iron overloaded in thalassemia patients gradually,also enhanced the risk of susceptibility to iron overload with thalassemia(OR=1.159,95%CI=0.797-1.359,P=0.553).Conclusions The study found that GDF-15 rs1058587 had GG and CG genotypes and based on CG genotype.On account of a greater likelihood of the limit of testing sample size and low extremely mutation frequency in Guangxi population,CC genotype was not found in this study.These data suggest that susceptibility to iron overload in thalassemia patients may be influenced by GDF-15 rs1059369 and rs4808793 polymorphism but not for rs4808793 polymorphism.CHAPTER Ⅱ Analysis of GDF-15 genotype,serum levels of GDF-15,EPO,Hepcidin and Iron MetabolismBackground and Objective GDF-15 is a regulatory factor in iron metabolism and generation of erythroid that can adjust Hepc in organism.The serum level of GDF-15 in thalassemia patients,especially in severe thalassemia patients,is apparently higher than normal people.That indicates the close connection in GDF-15 and the occurrence or progress of iron overload in thalassemia patients.However,the mechanism of GDF-15 in iron overload in thalassemia patients and the influence in prognosis of GDF-15 are still unclear.Besides,the relation of circulating levels and genotype of GDF-15 with iron overload in thalassemia patients remains indefinite while it’s crucial in stated functional mechanism of relation with lesion degree of iron overload in thalassemia patients.In this part of study,we analysed the level of GDF-15 in iron overload in thalassemia patients and the relation of that with iron metabolism to detect the association in GDF-15 and iron overload for adding new maker in diagnosing iron overload in thalassemia patients.Methods Patients group: selecting 284 thalassemia patients diagnosed in hematology and home of thalassemia in Guangxi Medical University from August 2016 to February 2017 of which included 122 iron overload-thalassemia patients,162 no-iron overload in thalassemia patients wherein included 123 males and 161 females,ages range from 0 to 60,68 dependency transfusion patients and 54 no-dependency transfusion patients.All of cases came from Guangxi as well as excluded other types of anemia,infectious disease,tumor,liver and bone disease history,etc.Controls group: 128 healthy controls came from First Affiliated Hospital of Guangxi Medical University in corresponding period of which included 54 males and 74 females,and people with anemia,inflammation and tumor were excluded.Inclusion criteria: person with serum ferritin and transferrin levels in normal reference interval and the normal routine inspection.Serum levels of GDF-15,EPO,Hepc,FER and Tr F were executed in all of samples included in our study and verified with WB。Results In this study,in three groups,statistically significant differences were shown in the serum levels of Hb,GDF-15,EPO,Hepc,FER,Tr F and Hepc/FER(P<0.05).We divided included population into dependency transfusion group and no-dependency transfusion group where in both of groups,statistically significant differences showed in the serum levels of Hb,GDF-15,EPO,Hepc,FER,Tr F and Hepc/FER(P<0.05).In the analysis of the association between the level of GDF-15 and Hb,EPO,Hepc,FER and Tr F,the serum level of GDF-15 in all the research objects were positively correlated with EPO,Hepc and FER(P<0.001)whereas that showed negative correlation with EPO,Hepc and FER(P<0.001).The serum level of GDF-15 increased sharply in thalassemia patients showed that no correlation with GDF-15rs1059369 and rs4808793 and the change of the allele frequency distribution,while showed negative correlation with Hb.Conclusions 1.The serum level of GDF-15 in thalassemia patients rised with the lowering of Hb,which implied the rising serum GDF-15 related with the anemia and oxygen deprivation.2.No correlation between the serum level of GDF-15 and GDF-15 rs1059369 and rs4808793.3.Comparing GDF-15 and Hepc,Hepc/FER is a more accurate and superior diagnosed maker in diagnosing iron overload-thalassemia patients.CHAPTER Ⅲ Analysis of glycosylation pattern of serous glycoprotein from iron overload-thalassemiaBackground and Objective Iron overload in thalassemia patients can contribute to multiple organs damage involved in the pathogenesis,therapeutic effect and progression of thalassemia.Glycosylation is a common posttranslational modification,which play an important role in cell adhesion,signal transduction,structural stability,receptor construction,cell differentiation and immune regulation.In this part of study,we used lectin microarray to detect differential glycan profiling of serous glycoprotein from iron overloadthalassemia patients.Here,we aim to screen specific biomarkers that related to diagnosis or activity of iron overload in thalassemia patients.Methods Experimental process was as followed:(1)Patients were divided into 4 groups,including iron overload-αthalassemia patients,no-iron overload inαthalassemia patients,iron overload-βthalassemia patients,no-iron overload in βthalassemia patients.Equal volume sample were mixed to acquire pooled serum.Low-abundance protein was collected using high-abundance protein removal kits.(2)After desalting,the low-abundance proteins were diluted with labeling buffer.(3)The target proteins were labeled with biotin using LightningLink kits.(4)Prepare the lectin microarray and hybridization.(5)Lectin microarray was scaned and data was collected.(6)Data was analysis by the microarray.SPSS 16.0,two independent samples t test or non-parametric test was used.(7)Agglutinin blotting technique to detect the affinity of AAL,LCA and WGA lectins to serum glycoproteins and verify the reliability of the results of lectinsResults A total of 34 lectin-positive affinity signals were screened out from the four group samples in this study,among them,19 differentiating lectins were screened out from α-thalassemia group and other 15 were screened out fromβ-thalassemia group.Compared with the non-iron overload group,α-thalassemia iron overload group showed increased lectins affinity in AAL,LCA,ABL,AMA,MNA-M,DBA,GHA,GSL1,GSL1b4,HAL,GSL2,GNL,PHA-L,PSA,SSA and LAL,while decreased lectins affinity in RCA60,STL and WGA.Compared with the non-iron overload group,β-thalassemia iron overlord group showed increased lectins affinity in ABL,GSL2,LCA,STL,TL,AMA,PSA,while decreased lectins affinity in Con A,GNL,NPL,IRA,HAL,HPL,DSA and MAL-II.Agglutinin blotting technique demonstrated that the results of AAL,LCA and WGA agglutinin affinity for serum glycoproteins were consistent with the results of lectins microarray.Results The tendency of various lectin affinity signals varied,indicate that in the progress of the thalassemia iron overload occurred,serum glycoprotein sugar chain structure of different types of thalassemia has complex and specific changes,suggesting that specific glycan patterns of serous glycoprote might be efficient biomarkers of diognosis or progression of thalassemia iron overload.