Clone And Polymorphism Research Of RHAG Gene

Rh blood group system is the most complex of the 30 blood group system found on human red blood cells,which is consisted of at least 45 antigens,and D is the most immunocompetent of all.The high antigenicity of Rh antigens makes this system of important clinic significance.Rh antigens are associated with transfusion actions, hemolytic disease of the new born or autoimmune anemia.RHAG is the 30^th human blood group system,which is consisted with 3 antigens at present and controlled by RHAG gene.RHAG and RHD belong to different blood group system,but they are related to each other closely.The D antigen of RHD blood group system is regulated by RHAG gene,the inactivity of RHAG could result in the deletion of all the Rh antigens.The two genes are highly homologous,and both belong to the human RH gene family. They are 75kb DNA which consisted of 10 exons,and the sequences between exon2-9 are highly similar,which suggests that RH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event.RHD maps on 1p36.13～p34.3,which encodes RhD protein;while RHAG maps on 6p11～p21.1 and encodes RhAG protein.RhAG protein participates in the form of Rh complex on red blood cells.The research indicated that the principal part of Rh complex is a trimer of RhD and RhAG, which also connects with CD47,LW,GPB and Band3,and form an Rh macrocomplex on RBCs. When RHD is deleted or some there is a mutation of RHD,the encoded amino acid changed,the structure and function of protein also changed,and the Rh antigen will change finally.RhAG don't have antigencity,but will impact the expression of RhD and RhCE.The mutation of RHAG will lead to the very rare Rh deficiency syndrome: Rh_null and Rh_mod,all the Rh antigens are absent on the RBCs of these types.There are two genetic background of Rh_null,one is the homozygous of RHD deletion and inactivity of RHCE;and the other is the homozygous of RHAG deletion. Some Rh_null are formed by one or two missense mutations of RHAG,which lead to the replacement of some amino acids,these tiny varieties make RHAG inactivity.An exhaustive study is made to the structure and function changes of Rh_null RBCs. The impact of Rh_null to antigens on RBCs is not only in Rh blood system.On Rh_null and Rh_mod RBCs,LW,GPB,CD47 and some other antigens are deleted or at least weak expression.With RT-PCR and sequencing,the RHD and RHCE cDNA of Rh_null RBCs are normal,which indicate that Rh_null is not arised from the mutation of RHD and RHCE.But the RT-PCR results of RHAG revealed that Rh_null RBCs are different from control RBCs.In these rare Rh_null individuals,RHAG mutated and stop RhAG, RhD and RhCE to connect with the membrane of RBC.This shows RhAG is very important for RhD to set up correctly on the membrane of RBC.The polymorphism of RHD is complicated,and there is a population difference. RHAG is more conservative than RHD,and only 13 alleles were found,which are all located on rare Rh_null and Rh_mod individuals.The RHD negative may have many genetic backgrounds,and the function of RHAG to it is still unknown.As to normal RhD phenotype,especially the expression of RHAG in Chinese population needs more investigation.So it is significant to clone RHAG in Chinese population, sequencing and analyze the polymorphism.Besides,in some RhD variants,RHD gene don't agree with RhD phenotype.We need to detect RHAG gene of these samples.There are successful reports about expressing RHD in K562 cells,but it is still difficult to clone and con-transfect RHD and RHAG in cell lines.And there is no report about the con-transfection RHD and RHAG of RHAG mutant.We chose normal RhD individuals and RhD variants as experiment samples,established the method of amplifying RHAG transcript and promoter sequence,construct pcDNA3.1⁺-RhAG and pcDNA3.1⁺-RhD vectors successfully,and establish the West-blot and Flow Cytometry methods to detect RhD and RhAG proteins.Finally we obtain the following conclusion:1.In the normal RhD individuals of Chinese population,there is no polymorphism of RHAG transcript,while the polymorphism exit in promoter sequence;2.While the polymorphism exit in promoter sequence of normal RhD individuals and RhD variants;3.A new RHAG mutation was found firstly in Chinese population,it is a 582 C＞T homozygosity mutation in RHAG gene.There is still no report about the investigation of RHAG transcript and promoter sequence in Mongolian,we explore the RHAG transcript and promoter sequence of normal RhD individuals and RhD variants in Chinese population.We find RHAG of RhD variants generally normal,and RHAG has little impact on the expression of RhD protein.As to the RHAG mutant,We still need to investigate the co-expression of RhD and RhAG.