Molecular Mechanism Of RhD Negative Phenotype In Chinese Han Population

The Rh blood group system is the most complex of all blood group systems with 49 antigens and numerous phenotypes. It is also the most clinically significant blood group except ABO system in blood transfusion medicine. The highly immunogenic RhD antigen is often involved in hemolytic disease of the fetal and newborn(HDFN), hemolytic transfusion reactions and autoimmune hemolytic anemia. Since the genes, RHD and RHCE, which encode the two Rh proteins were cloned in 1990s, the mechanism of Rh blood group polymorphism is greatly being known. Recently the immunological and molecular study of RhD antigen and RHD gene have made great progress. In 2000, RHD gene was found to be flanked by two highly homologous gene fragments, called Rhesus boxes and the mechanism of RHD gene deletion was firstly explored. The study of RhD antigens and numerous D variants(so far 40 weak D types and 25 partial D types have been found) showed the complex phenotype in serology results from complicated molecular basis, includeing single nucleotide polymorphisms(SNPs), gene deletion and insertion, gene crossing-over, gene rearrangement and gene conversion. Since the molecular mechanism of Rh blood group was clarified, the molecular methods, mainly PCR, were gradually applied in clinical medicine. RHD genotyping and fetal DNA determination in maternal plasma is of the most clinically significance. In addition, analysis of the RHD gene zygosity of husbands can predict the HDFN incidence of the fetal suffered. Genotyping can also solve the serological difficulties caused by repeat and large volume blood transfusion, D variants(weak D and partial D), etc.However, the study of RHD gene showed apparent racial difference. The genotyping application which based the Caucasian genetic RHD structure has limitation in other races. The RHDΨand RHD-CE-D~S gene were usually found in African and made false result of RHD genotyping. One extremely weak D called Del type was found in Asian people and this type covers 10-33% of Japanese and Chinese RhD negative donors. Del type individual has almost intact RHD gene and the weak D antigen of Del can only be detected by complicated absorb-elution test. Some other RHD-CE-D hybrid gene and small amount of gene mutaions were found in Asian RhD negatives. The study of RHD gene in Asian people(including Chinese) was greatly left behind than in that of Caucasian. It remains unclear in immunological and molecular mechanism of Del, RHD-CE-D hybrid gene, weak D and partial D in Asian. It's also unclear in Rhesus boxes gene sequence and genotyping in Rh syetem therefore can not be used rightly and reliably.In this stduy, the advanced molecular technique of real-time fluorescence quantitative PCR(RQ-PCR), multiplex PCR(MPX-PCR), PCR-restrict fragment lenth polymorphism(PCR-RFLP) and DNA sequencing were applied to systematically analyse and study the genetic structure of RHD gene in Chinese Han population. The aim of this study is to constitute data of Chinese RHD gene structure and futher to applying genotyping into clinical medicine.Objectives To analyse and study the genetic structure of RHD gene in RhD negative, RhD positive and partial D individuals and explore the molecular mechanism of D negativity in Chinese Han population. To analyse the PCR product of Rhesus boxes gene ampliation and determine the zygosity of RHD gene. To constitute data of Chinese RHD gene structure and futher to applying genotyping into clinical medicine. Materials and Methods DNA of 176 D-negative samples, 11 D-positive samples and 8 partial D samples was analysed by using an RHD multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by PCR-restriction fragment length polymorphism (PCR-RFLP) for RHD zygosity determination. Sixty-five samples were additionally analysed by using real-time quantitative PCR on RHD exon 7. RHD exon-specific sequencing was performed on discrepant samples. PCR products of 27 samples obtained with primers rez7 and rnb31 (amplifying both the downstream and the hybrid Rh boxes) were sequenced with consensus primer pairs CRBF06/CRBR08 and Rhbox5070/Rhbox5571 to determine the Rh boxes sequences. Results 114 samples (65%) showed the absence of RHD-specific exons by RHD MPX PCR and homozygous RHD negativity by PCR-RFLP. 49 samples (28%) which were defined as Del type in serological test showed the presence of RHD-specific exons by RHD MPX PCR and RHD gene positive by PCR-RFLP, 22 samples in this group showed a 1227G>A mutation, characteristic for the Del phenotype. 12 (6%)samples showed all characteristics of the RHD(1-2)-CE(3-9)-D(10) hybrid gene. One sample (1%) showed a novel 933C>A nonsense mutation in RHD exon 6, which resulted in a premature stop codon. Specific sequencing of the Rh boxes indicated that the physical structure of the hybrid Rh box on the RHD-negative RH locus in Chinese people is identical to the structure previously described in White people. 8 partial D samples showed the presence of 1,7,8,9,10 exons and absence of 3,4,5,6 exons of RHD gene. Conclusions The RHD gene deletion, RHD gene 1227G>A Del mutation, RHD-CE-D hybrid genes and one novel 933C>A mutation were found to be the four mechanisms that cause D negativity in our samples. The 1227G>A Del mutation was found to be the major cause of discrepant results between genotyping and phenotyping strategies, favouring genotyping of D negative samples. Sequencing of Rh boxes show that Chinese RHD gene deletion has taken place within the defined breakpoint region as in Caucasians. The partial D type in Chinese has special genetic structure of RHD gene and should be treated as RhD positive when donating blood.