Molecular Epjdemiological Survey Of Haemoglobinopathies In Greater Guangzhou Area And The Study Of Mechanism That ZBTB7A Coordinates With NuRD Complex To Increase HbF Level In Nondetional-HPFH

Haemoglobinopathies is one of the most common Mendelian genetic diseases in the world,including thalassemia,abnormal haemoglobin disease and HPFH.Thalassemia intermedia(TI),thalassemia major(TM),and some types of abnormal hemoglobin(hemoglobin variants)may cause severe clinical symptoms.There are no effective therapies for them,which make it become a global public health problem.Hemoglobin disease has obvious ethnic and regional differences.Mobility and migration of people affected the distribution of haemoglobinopathies.It has become the research focus of epidemiology of haemoglobin disorders to illustrate the difference of gene spectrum caused by migration,which gives reliable laboratory datas for prenatal diagnosis and genetic consultation.The magnitude of HbF elevation may relieve the symptoms of β-thalassemia intermedia or major.Point mutations or small deletions in the proximal fetal γ-globin gene promoters account for nondeletional HPFH.As one of the major fetal globin gene repressors,ZBTB7A was found to directly bound to this region which make it be a focus of hemoglobin research,but the regulation of ZBTB7A in γ-globin gene expressionIn this study,a total of 274,444 blood samples from 137,222 couples were obtained and analyzed in Guangzhou according to hematological parameters(MCV<82fL/MCH<27pg).A total of 137222 couples participated in pregestational thalassemia screening.74102 subjects from 37501 couples with MCV>82F1 or MCH<27pg were selected for further investigation.Gap-PCR,RDB,Sanger sequencing combined with Multiplex ligation dependent probe amplification(MLPA)was used for further study.Rare cases were analyzed by next-generation sequencing.We want to know the differences in the prevalence and molecular spectrum of eleven districts of Guangzhou and explore the effect of migrant populations for distributions of haemoglobinopathies.The correlation between the genotype and clinical phenotype was compared,which was useful for intervention for thalassemia.Several important experimental methods including shRNA,dual luciferase reporter assay,qPCR and so on will be used in K562 cells to explore the effect of the expression level of β and y-globin from ZBTB7A gene and NuRD complex,which were be useful for new gene target therapy.Results:1.There was a 11.34%and 0.38%prevalence of thalassemia and abnormal hemoglobin respectively in Guangzhou.The carrier rates of α-,β-,and combined α-/β-thalassemia were 7.76%,3.02%,and 0.5%,respectively.The differences between different districts were variable.The prevalence in the northern Guangzhou was much more than in the southern.Huadu district had a higher prevalence of thalassemia carriers but Nansha district had a lower prevalence(P<0.05).α-and β-thalassemia(α-and β-thal)gene mutation spectrum in the population of Nansha district was very different from other places.The gene composition ratio is unique,αQS is the third most common genotypes while the-28(A-G)gene ranked first.There are abundant types of abnormal hemoglobin in Guangzhou.Hb E,Hb New York,Hb Q-Thailand,and Hb G-Taipei are very common,which reflect the integration of population.Chinese Gγ(Aγδβ)0-thal,SEA-HPFH and the Taiwanese deletion are common genetic factors related to high HbF levels in Guangzhou.Deletion and mutation can cause the difference of hematological parameters.The number of genes involved is also one of the major factors.The carriers can be detected effectively using the appropriate gene detection method.Hematology parameters and the results of hemoglobin analysis should be considered.2.Aγ-196C>T mutation is very common nd-HPFH in Guangzhou.The blood indexes of Aγ-196C>T mutation carriers was normal.This mutation was in the site where ZBTB7A directly bound to.3.The results of double luciferase report experiment showed that over-epression of ZBTB7A gene significantly enhancing the luciferase activity of mutation-type promoter plasmid transfected cells.The double luciferase experiments also demonstrated that over-expression of NuRD complex(GATAD2B)inhibited the luciferase activity of wild-type promoter plasmid transfected cells slightly but enhanced the luciferase activity of mutation-type promoter plasmid transfected cells.When we inhibited ZBTB7A gene expression using shRNA in K562 cells,the expression of β globin increased significantly while that of y globin decreased significantly(P<0.01).Both of β globin and y globin elevated significantly in CHD3 knockdown K562 cells by CHD3-shRNA,espercially ZBTB7A gene knockdown at the same time.(P<0.001).This study is the first to describe the prevalence of haemoglobinopathies and the difference among 11 districts in Guangzhou.This study clarified for the first time that the gene carrying rate of hemoglobinosis in Guangzhou was higher in the south and lower in the north.There is different in the gene composition ratio from district to district,espercially in Nansha.The result of genetic testing should be consistent with the phenotype.Combined multi methods is helpful to detect carriers.Based on literatures,our experiments presented that ZBTB7A up-regulated y-globin gene expression by binding to LCR(locus control region)while down-regulated y-globin gene expression by binding to its promoter with NuRD complex,which provide a useful reference for in-depth studies on transcriptional regulation of y-globin gene expression in the future.