Study Of RHD Gene Zygosity And Molecular Biology Of D Variants In Zhejiang Han Population

The Rh blood group system is one of the most significant red blood cell systems in transfusion medicine, incompatible Rh antigens are associated with hemolytic transfusion reactions, hemolytic disease of the newborn and autoimmune hemolytic anemia. The common Rh antigens are D, C, c, E and e, antigens of the Rh blood group system are products of RHD and RHCE, two tightly linked and highly homologous genes that were organized on chromosome Ip34.1-lp36. Individuals are classified as either RhD positive or RhD negative basing on the presence or absence of the D antigen on the red cell surface.Two 9kb highly homologous sequences are located at the 5' and 3' ends of RHD, which were named "upstream Rhesus Boxes" and "downstream Rhesus boxes" . There is an overall 98.6% homology between both Rhesus boxes, me orientation of two Rhesus boxes is identical to RHD. In Caucasians population, the RhD negative phenotype is usually existed a deletion of the full RHD gene that had occurred by an unequal crossing-over triggered by the highly homologous Rhesus boxes embracing the RHD gene, leaving only a single hybrid Rhesus box. The 5' end of this hybrid Rhesus box represented an upstream Rhesus box and the 3' end represented a downstream Rhesus box, the full of RHD gene was absence between upstream Rhesus Boxes and downstream Rhesus boxes and formed the new hybrid Rhesus box.RHD zygosity was spectulated by Rh phenotype, quantify of RHD gene, the presence of the neighboring gene of RHD in the past few years. After identified the Rhesus box and elucidated the genetic structure of RhD phenotype in Caucasians, PCR with restriction fragment length polymorphism (PCR-RFLP), doubleAmplification Refractory Mutation System, real-time quantitative PCR and PCR with sequence specific primer (PCR-SSP) were used for detection of the RHD zygosity in individuals. It is important of determination RHD zygosity for individuals, which can be used for assessment the risk of HND by maternal anti-D isoimmunization and prediction the RhD phenotype of the newborn. In D-negative mother, the child is 100% RhD positive with an RHD+/RHD~+ father, but it is only 50% possibility RhD positive with an RHD~+/RHD~- father.The aims of this study were to determinate RHD gene zygosity in Zhejiang Han population, identify the molecular basis of D variants in Chinese and detect the 1227G>A mutation in RhD negative individuals and the 845G>A mutation in RhD negative individuals and in D variants individuals. We used the PCR-SSP method to detect upstream, downstream and hybrid Rhesus box and speculated the gene zygosity of the individuals according to the Rhesus box pattern. And we amplified RHD exons and its flanking regions, the amplification products were directly sequenced to determine the mutations of D variants. We also used the PCR-SSP method to detect the 1227G>A mutation and 845G>A mutation.The results showed that only hybrid box could be determined in RHD'I RHD' homozygosity. All the upstream box, downstream box and hybrid box could be determined in RHD* I RHD' heterozygosity, while upstream box and downstream box except hybrid box could be determined in RHD+/RHD+ homozygosity. 12 (6.06%) were RHD+/RHD' heterozygosity and 186 (93.94%) were RHD+/RHD+ homozygosity in the 198 RhD positive samples. In 32 D variants, 29 (93.63%) were RHD*/RHD' heterozygosity and 3 (9.37%) were RHD+/RHD+ homozygosity. In 208 RhD negative samples, 53 (25.48%) were RHD+/RHD~ heterozygosity, 145 (69.71%) were RHD'/RHD' homozygosity, 10 (4.81%) were RHD+/RHD+ homozygosity. Using the direct RHD gene sequencing method, five with 845G>A mutation, threewith 1227G>A mutation, and one with 1013T>C mutation were characterized among nine weak D phenotypes. One Dva (Kou.), one Dva (Hus.), one DVa-like (YH.) and seven DVI type III were characterized among ten partial D phenotypes respectively. In 208 RhD negative samples, 39(18.75%) were detect with 1227G>A mutation;2 (0.96%) were detect with 845G>A mutation. In 32 D variants, 12(37.5%) were detect with 845G>A mutation.It is concluded that the distribution of RHD gene zygosity is different among RhD positive, RhD negative and weak D in Zhejiang Han population. There are many RHD+IRHD~ heterozygosity and RHD+/RHD+ homozygosity in RhD negative phenotype, which are suggested that there are partial RHD gene deletion and whole RHD gene existence in Han population with RhD negative phenotype. A sequence method for RHD gene was established and the molecular basis of nine weak D and ten partial D phenotypes were characterized. Dva (Kou.) and DVa-like (YH.) phenotypes were first reported in Chinese population.