Study On RHD,RHCE Genes And Transcriptional Diversity

BackgroundThe Rhblood group system is one of the most polymorphic human red blood cell blood groups,and 5 antigens,D,C,C,e,and E,are clinically important,including D antigen,which is the most immunogenic of the Rhblood group antigens,and the high immunogenicity of the RhD antigen often causes hemolytic disease of the newborn and hemolytic transfusion reactions.Rhantigens are encoded by the RHD or RHCE genes,and the mechanisms involved in the formation of polymorphisms between RHD and RHCE genes include gene deletions or insertions,unequal gene exchange,splice site mutations,gene recombination,and gene hybridization,among others.A subset of RH variants do not have mutations in the coding region but have altered protein expression levels,suggesting that the mechanism may be related to polymorphisms in noncoding regions as well as alternative splicing and that aberrant mRNA splicing and intron retention may affect D antigen expression.The analysis of RHD gene expression and splicing isomers is one of the hotspots and difficulties in the study of Rhblood group system.The technical means used mainly include q PCR,first-generation sequencing and second-generation sequencing.The third-generation sequencing technology emerging in recent years has realized the long-chain single-molecule sequencing,promoted the related research of homologous gene isomers,and was able to find new transcripts and quantitative,alternative splicing and other structural variations,which may bring more abundant information for related research.In this study,the transcripts of RHD and RHCE genes in RhD-positive and Del-type individuals were studied by using the Nanopore long reading sequencing technology,to understand the differences between the two phenotypes and their encoded proteins,and to carry out the relative quantification of various splices.Objective1.Knowledge of the genetic background of RhD Negative blood donors by RHD and RHCE genotyping.2.To establish a method for sequencing and quantification of RHD and RHCE gene mRNA transcripts using single-molecule nanopore long read three generation sequencing to provide a new method for mRNA alternative splicing studies of blood group gene variants.3.To explore the species and expression level differences of mRNA transcripts between RHD and RHCE genes.Methods1.RHD and RHCE genotypingFrom March 2021 to December 2022,56 EDTA anticoagulant samples and 15 RhD positive samples,which were negative in the RhD preliminary screening and confirmatory test,were sent to our laboratory by hospitals in Chengdu to prepare DNA and detect the purity and concentration with Nano Drop2000.RHD and RHCE genes were amplified by sequence-specific primer PCR reaction for genotyping.RHD gene deletion was identified by detecting upstream box（UP）and hybrid box（HY）.After amplification,it was imaged and analyzed after 20 min of 110 V electrophoresis on1.5%gel.2.RHD and RHCE gene c DNA amplification and Sanger sequencing verificationThe buffy coat and nucleated red blood cells were separated by Ficoll density gradient centrifugation from the variant samples with genotype Del 1227G>A and RhD positive samples（within 48 hours of collection）.After washing,1m L TRIZOL was added and fully mixed.The RNA was extracted with chloroform and isopropanol and reversely transcribed into c DNA.Nano Drop2000 was used to detect RNA purity and concentration.Using Primer Premier6 software to design a pair of primers,RHD and RHCE c DNA can be amplified at the same time,and the amplification is completed by 110 V electrophoresis on 1.5%gel for 30 minutes before imaging.The target band was cut under ultraviolet light and purified and sequenced by Sanger.The sequencing results were compared with the standard sequence of Genebank using Snap Gene software,and the amplified target gene was verified in BLAST of NCBI.3.Research on splicing isomers of RHD and RHCE c DNA based on NanoporeFive Del samples and five RhD positive samples with good concentration and purity were selected to amplify RHD and RHCE genes.The PCR products were purified with exonuclease and quantified with Qubit fluorometer.The SQK-LSK109 kit from Nanopore was used to connect each sample with DNA end repair,A-tail and Barcode tag,and then the library was collected,and sequencing connectors were added.Finally,AMPure XP magnetic beads purification was completed to complete the library construction.The whole full-length transcriptome sequencing is controlled and monitored by Min KNOW software.Sequencing data is analyzed by using the Bioinformatics software,including sample barcode splitting,filter quality control,quantitative TPM of each transcript of each sample,variable splicing analysis,etc.4.Study on the difference of protein encoded by splicing isomersTranslate the c DNA sequence obtained from the third generation sequencing into amino acid sequence,and use Bio Edit software to compare the amino acid sequence of each transcript with the normal RhD protein sequence for the primary structure.Deep TMHMM was used to predict the transmembrane structure of the target gene protein,and TOPO2 tool was used to draw the transmembrane structure pattern map.Using the idea of homology modeling,the normal RhD protein and the amino acid sequences translated from each splice were imported into the Robetta homology modeling website to build the 3D conformational model of each RhD splice protein.Results1.RHD and RHCE genotypingCombined with the detection results of 10 exons and upstream and downstream boxes,13 of the 56 RhD negative samples were heterozygous with complete deletion of RHD（1227G>A）and 1-10 exons,2 were only heterozygous with complete deletion of RHD（1227G>A）,1 was heterozygous with complete deletion of RHD-RHCE（4-9）-RHD and 1-10 exons,and the remaining 40 samples were true negative,and 10 exons（d/d）of RHD gene were completely deleted.RHCE genotyping results showed that 18 cases of RhD negative were Ccee（32.1%）,32 cases were ccee（57.1%）,3 cases were CCee（3.5%）,2 cases were cc Ee（3.5%）,and 1 case was Cc Ee（1.9%）.2.RHD and RHCE gene c DNA amplification and Sanger sequencing verificationThe OD_260/280of RNA in all samples was between 1.7 and 2.1.After reverse transcription into c DNA and amplification of RHD and RHCE gene transcripts,the electrophoresis results of samples with different Rhgenotypes showed multiple clear bands of different lengths.The sequences obtained by Sanger sequencing after strip gel cutting purification were all transcripts of RHD or RHCE genes after BLAST query.3.Research on splicing isomers of RHD and RHCE c DNA based on NanoporeTen RHD mRNA transcripts and nine RHCE mRNA transcripts were detected in 10samples.There is a full-length transcript of RHD（RHD-201）in Del type,but the expression level is significantly lower than that in RhD positive samples（P<0.001）.The expression level of transcript RHD-207（Del789）in Del type is significantly higher than that in RhD positive samples（P<0.05）.The transcript RHD-208（Del8910+213）is only detected in Del type individuals（P<0.05）.There is no significant difference between other RHD transcripts and all RHCE transcripts in the two individuals.RHD gene has 8 exon jumps,2 mutually exclusive exon splicing,and 3 last exon splicing,which all occur on the positive chain.RHCE gene has 6 exon jumps,1mutually exclusive exon,and 7 variable splicing events,all of which occur on the negative chain.There was no statistical difference in the probability of RHD and RHCE variable splicing events between RhD positive and Del type（P>0.05）.4.Study on the difference of protein encoded by splicing isomersBioEdit analysis showed that the 10 splicing bodies of RHD gene formed a total of8 proteins,with differences in glycine at the 314th position.The protein encoded by RhD full-length transcript crosses the membrane 12 times,and the differential protein part of all splicing proteins is located in the cell membrane.The 3D structure model of the protein showed that the final amino acid of the splicing RhD protein had different degrees of helical conformation changes.Conclusion1.This study has successfully established an analytical method for sequencing the full-length transcript isomers of RHD and RHCE gene mRNA in three generations,which can detect the transcripts with low abundance and quantify each alternative splicing transcript.2.RHD1227G>A variant can normally transcribe the mRNA containing exon 9,but most of them cut out exon 7～9 during the splicing and maturation of the mRNA,and only a few form full-length transcripts.3.This study found that only the RHD-208（Del8910+213）transcript exists in Del samples,which belongs to the intron retention transcript of non-translated amino acids.There have been few reports before.The alternative splicing of the non-coding region of the gene may cause the decrease of RhD antigen expression,and the relationship with1227A Del type needs further study.4.RHD and RHCE genes have complex variable splicing.There is no difference between transcripts of different RHCE genotypes.The transcription and splicing of RHCE genes may be relatively independent fromRHD and RHCE genotypes.