Identification Of Two Novel Large Segmental Deletion Involving ?-globin Gene Cluster Causing Thalassemia

Background and objectivesAlpha-thalassemia is an autosomal recessive inherited disease,mainly manifested as small cell hypochromic anemia.The molecular basis of alpha thalassaemia major is mainly deletion mutation and a few point mutations,which reduces the production of alpha-globin protein.Too much gamma globin chains is formed to Hb Bart's(?4)in fetus and too much beta globin chains is formed to Hb H(?4)in adult,resulting in ineffective hematopoiesis and destruction of red blood cells.Expression of a genes is dependent on remote regulatory elements(multispecies conserved sequences or MCS-R)located 30-70 kb upstream of the a genes within a flanking.Among them,MCS-R2(HS-40)is a powerful erythroid-specific enhancer and its core element,representing a fragment with length of 258 bp,contains several binding sites of erythroid-specific transcriptional factors,in particular GATA1/2 and NF-E2.The purpose of this study was to identify a family with large fragment deletion of the alpha-globin gene cluster and a family with large fragment deletion of the distal regulatory element of the alpha-globin gene(including HS-40).Both of these deletion mutations reduced the expression of alpha-globin gene and aggravated the patient's clinical phenotype.Materials and methods:1.Research object:the proband of family A is a 29-year-old man from Liuzhou,Guangxi.The hematologic parameter is:MCV 56.4fL,MCH 17.8pg,Hb H 6.6%,manifested as Hb H disease.The proband of family B is the Hb Bart's hydrops fetalis syndrome fetus from Liuzhou,Guangxi.2.Experimental methods:to analyze the hematological phenotype of the proband and its family members,and carry out the conventional mutation detection(Gap-PCR and RDB)combined with the results of phenotypic analysis.The results of common mutation detection were not sufficient to explain the phenotype of the proband,so the CNVs of the alpha-globin gene cluster and the point mutation of HBA and HBB genes were detected by MLPA and Sanger sequencing respectively.The results showed that in addition to the-a3'7/deletion,the proband of family A had a large segmental deletion that included two a-globin genes.In addition to the--SEA/deletion,the proband of family B had a large fragment deletion that included the distal regulatory element of a-globin genes.Because the deletion location of family B is beyond the range of MLPA probe,we used NGS technology to provide targets for more precise breakpoint localization.According to the breakpoint shown by the NGS,we used the Gap-PCR method with specific primers at both ends of the breakpoint to characterize the deletion breakpoint.Research resultsThe genotype of the proband in family A was Hb H disease,and the results of Gap-PCR?MLPA and NGS showed that the genotype was-?3.7/deletion compound a novel large segmental deletion involving a-globin gene cluster.Through family analysis,it was found that-a3'7/deletion was inherited from the father and the deletion of a novel large segmental deletion was inherited from the mother.Through the identification of breakpoint,it was determined that the deletion range was chr16:197578-257871,and the deletion size was 60295bp,and the genotype of the proband was--3.7/--60.The results of the hemoglobin electrophoresis of the proband in family B were Hb Bart's 88%and Hb Portland 11%and Gap-PCR?MLPA and NGS showed that its Genotype is--SEA/deletion compound a novel large segmental deletion containing HS-40.Similarly,the family analysis were found that the--SEA/deletion was inherited from the mother,and the novel large segmental deletion was inherited from the father.In addition,through the identification of breakpoint,it was determined that the deletion range was chr16:96622-176317,and the deletion size was 79696bp,and the genotype of the proband was(??)Lz/--SEA.HS-40 deletion on this chromosome leading to decrease two alpha-globin gene expression,which can function as--SEA/deletion mutation and explains that the proband is a serious Hb Bart's edema fetus.ConclusionThis study identified a novel large fragment deletion involving alpha-globin gene cluster and a novel large fragment deletion including distal regulatory sequence HS ?40.Both of the deletion mutations aggravated clinical phenotype,especially the HS-40 deletion,which severely reduced the expression of alpha-globin gene.These two cases enriched the alpha-thalassemia mutation spectrum,and re-confirmed that HS-40 is a powerful enhancer for the expression of alpha-globin gene in the population.