| BackgroundRh blood group system is one of the most important blood group systems in human and has very important clinical significance.In addition to the normal phenotype,there are also many variants of Rh blood group,related individuals can produce homoantibodies,leading to hemolytic transfusion reaction(HTR)and hemolytic disease of the fetus and newborn(HDFN),etc.RhD genotyping of RhD variants has been studied systematically and early in the Caucasian crowd and directed clinical blood use.In recent years,several qualified blood centers or laboratories in China have carried out studies on RhD variants in The Chinese population.However,China is a multi-ethnic country with a rich genetic background.Therefore,systematic research data on RhD variants in the Chinese population need to be improved.Previous studies have shown that most RhD variants are caused by mutations of RhD and RHCE encoding genes in the Rh blood group system,and are mainly related to D antigen,including partial D,weak D and D-elute(Del).Weak D15 and DVI.3 are the most common RHD alleles in Han Chinese population,accounting for more than 65%of the reported RHD alleles in China.With the development of molecular biology and the application of new detection techniques,more and more new RHD variant alleles will be discovered in the Chinese population.The purpose of this study was to explore the molecular biological characteristics of RhD variants in Guangzhou.By constructing an in vitro expression system,the expression characteristics and regulatory mechanism of RHD alleles can be effectively elucidated,and to explore the causal relationship between RHD alleles and D antigen phenotypes.To provide a basis for elucidating the molecular basis of the blood group of Rh variants in the Chinese population and establish corresponding genetic testing and safe blood transfusion strategies.Object1.To explore the epitope characteristics of individual D antigens of RhD variants in Guangzhou and the genetic molecular mechanism of gene mutation,and to understand the type distribution of RhD variants in this area.2.The in vitro expression system of 293T cells(human embryonic kidney cells)for the study of RHD allele D antigen was constructed.To clarify the molecular basis of Rh variant individual blood group in the local population,establish the corresponding gene detection and safe blood transfusion strategy.Methods1.From January to August 2019,RhD variant individuals detected in voluntary blood donors in Guangzhou and medical institutions in the region were sent to the central laboratory for Rh intractable blood group identification and confirmed as RhD variant after serological screening.59 blood samples from patients with genotype;in vitro expression study of a new RhD variant(RHD*497G)allele discovered by the research group earlier.2.Serological characteristics of D epitopes were further analyzed by using two monoclonal ants-D reagents and D-screen epitope detection kits,and RhCE phenotypic typing was performed by typing cards;QuickGene DNA extraction kit was used to extract the genomic DNA of the samples,and PCR-RFLP method was used to analyze the RHD gene zygote type;The RHD gene sequence was detected by Multiple ligation-dependent probe amplification(MLPA)genotyping,and the RHD exon(1～10)Sanger sequencing was performed on the samples still in doubt after the above detection,and the results were analyzed by DNAStar/SeqMan software.3.The target gene(RHD*497G)plasmid was constructed and 293T cells were cultured.The target DNA(RHD*497G)was transfected into 293T(human embryonic kidney cells)using FuGENE(?)6 transfection reagent for in vitro expression.A series of monoclonal antibodies against different RhD epitopes were used to detect the expression of D epitopes by flow cytometry.Results1.In this group of RhD variants in Guangzhou,27.12%(16/59)were detected from blood donors[accounting for 0.007%(16/232 793)of blood donors in Guangzhou during the same period].The difficult specimens sent by the hospital accounted for 72.88%(43/59).The main phenotypes of 59 RhD variant RhCE cases were 39%(23/59)Ccee,35.6%(21/59)ccEe,and 25.4%(15/59)CcEe.2.RHD genotype detection:40.7%(24/59)are RHD*weak partial 15,25.4%(15/59)were RHD*DVI.3 and 33.9%(20/59)other less common RHD variant types[76.92%(10/13)were RhD variants with 2 different alleles];Serology showed that DVI.3 cells and the nine mAbs in the D-screen kit showed a relatively fixed pattern and the remaining RhD variants can not be clearly identified by serology.3.In this study,there were 10 cases of RHD+/RHD+ homozygous(16.95%)and 49 cases of RHD+/RHD-heterozygous(83.05%),indicating a high rate of deletion of RhD variant allele in this region.In other words,the RHD+/RHD-allele was the main gene combination type of RhD variants in this region.4.The target DNA(RHD*497G)was transfected into 293T(human renal epithelial cells),and the antigens of RHD genethe were expressed by cultured cells,and then the results were detected by flow cytometry;The detection results of monoclonal anti-D clone number(antigen epitope)were as follows:5C8(2.2):+++,21G6(9.1):+++,brad-8(2.2):++,FOG-1(6.7):+++,D59897:+++,H41(3.1):+++,HIRO-4(9.1):+++,HIRO-5:+,HIRO-9(6.3):+++,LHM76/58(8.1):+++,LHM50/2B(6.3):++,LHM76/55(3.1):+++,LHM76/59(15.1):+++,Los-1(6.3):++,Los-2:++,MmABS a-D:+++,MS26(9.1):+++,poly a-D:+++,P3×241(5.4)and Brad-5(6.8)were not expressed.Conclusion1.Weak D15 and DVI.3 are the most common RhD variants in Guangzhou Han population.DVI can be preliminarily identified by serological methods such as D-screen anti-D reagents,while other RhD variants can be identified only by molecular biological methods.100%of the D variants in this study contained C or E antigens.2.The variant(RHD*497G)was successfully expressed by creating in vitro expression system and the results showed that some D antigen phenotypes were consistent with the serological results.The intensity or deletion of corresponding different antigens were detected by flow cytometry,which more objectively confirmed the molecular basis of abnormal expression of D antigen epitope in erythrocytes of corresponding proband and had important scientific significance.It provides the basis for transfusion guidelines and pregnancy monitoring strategies of RhD variant individuals. |
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