Background Accurate typing of the D antigen is critical to provide guidance for blood transfusion and safe management of Rh-incompatible pregnancies. RHD genotyping assays have been widely adopted to improve D variant detection, especially for common RHD alleles with missense variants and RHD-CE-D hybrids. Few structural variants of the RHD gene are known to involve large fragments of non-RH sequences, despite causing phenotype-genotype discrepancies. Methods We had previously detected reduced copy numbers of RHD exon 10 in Chinese Han. Their RHD genes were further analyzed using long-read sequencing of RHD and whole-genome long-read sequencing using PacBio platform. The population frequency and practical relevance were evaluated, such as effects on D antigen expression and accuracy of routine RHD genotyping assays. Results A novel structural variant of the RHD gene, dubbed RHD-CE-TMEM50A-D, had distinct features: a short deletion of the non-coding region of RHD exon 10 (688 bp del), a large insertion (21.8 kb ins) involving the inversion of non-coding region of RHCE exon 10 (174 bp) and exon 7 to intron 2 of TMEM50A gene (21,648 bp), and several single nucleotide substitutions. The large delins fragment was firstly found in 9 individuals, further representing 4.1% of random D+ donors (40/982), 2.9% of D-C+/E+ individuals who carry the non-functional RHD*01N.04 (6/205), and 3.4% of weak/partial D individuals (7/207) who carry RHD*DFR2 and RHD*DVI.3, but 0% in a different cohort of Asian-type DEL individuals (0/205). The large delins fragment located in non-coding region of RHD exon 10 of four different alleles did not significantly change the D antigen expression but caused inconclusive results in several routine RHD genotyping assays. Conclusions The novel RHD-CE-TMEM50A-D allele represented approximately 1 in 49 chromosomes among the southern Chinese Han population. The variations of these RHD alleles involve the replacement of some RHD-specific sequences by the corresponding RHCE-specific sequences in the non-coding region of RHD exon 10, commonly used for RHD-specific primers design in genotyping assay. Hence, red cell genotyping assays, if applied to individuals of East Asian heritage, should recognize this relatively common allele to avoid RHD allele dropout.

[Objective] To establish a method for the genotyping of fetal M blood group antigen by extracting cell-free fetal DNA (cff-DNA) from maternal plasma, so as to guide the management of M antigen-negative pregnant women with IgG anti-M antibody during pregnancy. [Methods] A realtime fluorescent quantitative PCR (realtime PCR) method was established. The specificity and sensitivity of the method were validated by dilution of genomic DNA. Subsequently, a total of 12 M antigen-negative pregnant women were enrolled. The cff-DNA was extracted from maternal plasma, and fetal M antigen genotyping was performed by realtime PCR. Fetuses were classified as M-positive or M-negative according to the presence or absence of amplification curve. The accuracy of the method was validated by comparing fetal M antigen genotyping results with the serological results using the cord or peripheral blood of the neonate at birth. [Results] Among the 12 M antigen-negative pregnant women, anti-M was detected in five cases, of which four cases had IgG anti-M, and one case had fetal anemia. The results of fetal M antigen genotyping showed that 9 cases were M-positive (9/12, 75%) and 3 cases were M-negative (3/12, 25%). Serological results of blood samples collected after birth from four M-positive fetuses and one M-negative fetus were consistent with the genotyping results. [Conclusion] We have, for the first time, established a non-invasive prenatal genotyping method for fetal M antigen using maternal plasma cff-DNA, and preliminarily demonstrated the feasibility of this method.

[Objective] To conduct screening for rare blood types within important blood group systems for the Chinese population, such as Rh, Duffy, Kidd, P1Pk, Diego, and MNS, in the Guangzhou region, and to establish a corresponding rare blood type database and physical repository. [Methods] The saline medium microplate method was used to screen blood donors with the ccDEE phenotype combined with either Jk(a-) or Jk(b-). The polybrene microplate method was employed to screen for donors with Fy(a-), s(-), Lu(b-), Di(b-), k(-), and p phenotypes. The urea lysis microplate method was applied to screen for the Jk(a-b-) phenotype. A high-resolution melting (HRM) curve method was established for screening some donors with the Di(b-) phenotype. Subsequently, expanded phenotyping of antigens in the Rh, Kidd, MNS, Duffy, P1Pk, Lewis, Kell, and Lutheran blood group systems was performed on identified rare blood type donors using monoclonal antibodies. The test results are entered into the Rare Blood Type Bank Management System of the Guangzhou Blood Center, enabling functions such as confirmation reminders and cryopreservation storage when the donor donates again. Red blood cells of rare blood types are processed into frozen red blood cells for long-term storage. [Results] Among voluntary blood donors, 16 cases of the ccDEE combined with Jk(a-) phenotype were identified (0.221 7%, 16/7 216); 10 cases of the ccDEE combined with Jk(b-) phenotype (0.138 6%, 10/7 216); 78 cases of the Fy(a-) phenotype (0.169 5%, 78/46 012); 39 cases of the Lu(b-) phenotype (0.138 2%, 39/28 214); 31 cases of the s(-) phenotype (0.081 8%, 31/37 913); 22 cases of the Di(b-) phenotype (0.029 9%, 22/73 691); 30 cases of the Jk(a-b-) phenotype (0.010 1%, 30/298 250); and 1 case of the k(-) phenotype (0.001 3%, 1/77 382), which was further identified as KELnull phenotype (K0). No p phenotype donors were identified (0/88 528). A total of 228 units of frozen red blood cells were prepared. The screening results were compared and analyzed with rare blood type data from other regions. [Conclusion] This study, through a combination of different screening methods, significantly improved the efficiency of rare blood type screening while remaining cost-effective. By conducting large-scale screening and performing data informatization processing, a database and physical repository of rare blood types in the Guangzhou region were successfully established. This provides a strong guarantee for the timely supply of blood to patients with difficult-to-match and rare blood types in the region, effectively enhances the level of transfusion safety in the region, and offers a practical paradigm for constructing a comprehensive blood transfusion support system.

Alloanti-D is still one of the most common causes of severe hemolytic disease of the fetus and newborn in China, as rhesus immunoglobulin (RhIG) prophylaxis is not a routine practice throughout China. HLA plays an important role in the susceptibility to alloimmunisation against red blood cell antigens. This study was designed to identify susceptible and protective HLA alleles for alloanti-D immunisation after pregnancy in the southern Chinese D-negative (D-) pregnant women. In this study, a cohort of 116 true D- pregnant females who had not received prophylactic RhIG prophylaxis, had two or more pregnancies, and did not produce alloanti-D (non-responders group), and 122 true D- pregnant women with alloanti-D immunisation (D responders group), were enrolled. HLA genotyping (HLA-A, -B, -C, -DRB1, -DPA1, -DPB1, -DQA1, and -DQB1) was performed by third generation sequencing with nanopore technology. The phenotypic frequencies of HLA alleles were compared between the D responders group and non-responders group. The results showed that the phenotypic frequencies of HLA-DRB1*08:03 and HLA-DQA1*01:03 alleles in the D responders group were significantly lower than those in the non-responders group: 1.7% versus 13.1% [Odds Ratio (OR): 0.116, 95% CI: 0.026-0.518; pc = 0.029] for HLA-DRB1*08:03 allele, and 5.2% versus 18.8% (OR: 0.235, 95% CI: 0.092-0.600, pc = 0.019) for HLA-DQA1*01:03 allele. Our findings indicated that the presence of HLA-DRB1*08:03 or HLA-DQA1*01:03 alleles can be considered as a protective factor for alloanti-D immunisation in the southern Chinese D- pregnant women.

BACKGROUND:The s antigen expression is mainly determined by a single nucleotide polymorphism at c.143C (p.Thr48) on the exon 4 of GYPB gene. Several mutations on the GYPB gene have been reported to cause aberrant s antigen expression. GP.Mur has an extra 31-amino acid insertion encoded by the active compound GYP(B-A) exon 3, which closely locates at the upstream of p.Thr48. It has been reported to cause altered s antigen expression. MATERIALS AND METHODS:Serologic testing and flow cytometry analysis were performed to detect s antigen expression on RBCs of GP(B-A-B) hybrid glycophorins, including GP.Mur, GP.Bun and GP.HF. Several mutant plasmids based on the different sites between GYPB and GYP(B-A-B) alleles were constructed and transfected into HEK293T cells for in vitro expression, to reveal the key amino acids for the aberrant s antigen expression. RESULTS:Serologic testing and flow cytometry assay showed the RBCs of GP.Mur homozygotes reacted positively with IgG anti-s (P3YAN3) but negatively with IgM anti-s (P3BER). Flow cytometry analysis also revealed half level of s antigen expressed on the RBCs of GP.Mur, GP.Bun and GP.HF heterozygotes with ss genotype compared to S-s+ controls when detected by IgM anti-s (P3BER). Furthermore, in vitro expression study showed that p.Asn45 is critical for the epitope expression of s antigen detected by IgM anti-s (P3BER). DISCUSSION:The results demonstrated partial s antigen expression on GP(B-A-B) RBCs. In addition to p.Thr48, p.Asn45 is also important for the epitope expression of s antigen detected by IgM anti-s (P3BER). To avoid false negative serologic typing, it is recommended to use several different clones of monoclonal anti-s for the correct s typing, especially in the regions with high frequency distribution of GP(B-A-B) hybrid glycophorins.

BACKGROUND:D-- is a rare phenotype lacking the expression of the C, c, E, and e antigens and several high-frequency antigens on the red cells. Anti-Rh17 (Hr0) could be developed in individuals with the D-- phenotype to cause hemolytic transfusion reactions (HTR) and hemolytic disease of the fetus and newborn (HDFN). Nuleotide(s) change of the RHCE gene and RHCE-D-CE hybrid alleles are the common molecular basis of the D-- phenotype. STUDY DESIGN AND METHODS:One D-- Chinese patient detected in routine RhD and RhCE serologic testing and another D-- Chinese patient identified with anti-Rh17 were recruited. Further RHD, RHCE, and RHAG whole gene sequences were analyzed using the PacBio sequencing. A dual-luciferase reporter assay was performed to verify the effect of the variant identified in the promoter of the RHCE gene on the transcriptional activity of the reporter gene in vitro. RESULTS:A homozygous RHCE*Ce(1-111G)/Ce(1-111G) genotype and a heterozygous RHCE*CeN.08/Ce(1-111G) genotype carried one novel variant (c.1-111A>G) located in the GATA-1 motif of the RHCE proximal promoter was identified in two D-- patients, respectively. In the reporter assay, the luciferase transcriptional activity of the mutant RHCE promoter [c.1-111G] construct reduced from ~1.0 to 0.28 relative luciferase activity normalized to RHCE wild-type, with a ~72% reduction rate. CONCLUSION:The novel variant of the GATA-1 motif of the RHCE proximal promoter was identified to diminish the binding of the GATA-1 transcription factor and markedly down-regulate the transcription activity of the RHCE gene to abolish the expression of RhCE antigens, causing the rare D-- phenotype.

[Objective] To investigate the distribution of GP (B-A-B) hybrid glycophorins in several Chinese minority populations from southern regions of China (Guangdong & Guizhou). [Methods] Whole blood samples were collected from 536 blood donors representing 15 different Chinese ethnic minority groups, including She, Bouyei, Yi and Miao, as well as Chuanqing populations. Genomic DNA was extracted and GYP (B-A-B) genotyping was conducted by high resolution melting (HRM) minority method using the GYPB pseudoexon 3-specific primers. Direct sequencing of GYPB pseudoexon 3 was performed in the samples with variant curves. [Results] Only one genotype of GP (B-A-B) hybrid glycophorins (GYP*Mur/GYPB) was identified among these 536 samples. In total, 15 She (15/162, 9.26%), 18 Bouyei (18/113, 15.93%), 3 Yi (3/79, 3.80%), 3 Chuanqing (3/45, 6.67%), 2 Bai (2/42, 4.76%), 3 Miao (3/40, 7.50%), 1 Shui (1/12, 8.33%), 2 Gelao (2/12, 16.67%), 1 Tujia (1/8, 12.50%) and 1 Dong (1/6, 16.67%) blood donors with heterozygous GYP*Mur allele were identified. Among 8 Hui, 5 Manchu, 2 Mongolian, 1 Yao and 1 Li donors, no GYP (B-A-B) hybrid gene carrier was found. In addition, four nucleotide polymorphisms (SNPs) were identified in 6 samples with a variant melting curve detected by HRM. [Conclusion] GP. Mur is the most common type of GP (B-A-B) hybrid glycophorins among Chinese minority populations, with frequency varying across different populations. It is recommended to involve GP. Mur reagent cells in the antibody screening cells for populations with a high frequency of GYP*Mur allele.

HLA-DPB1*1781:01 differs from HLA-DPB1*03:01:01:01 by one nonsynonymous nucleotide substitution in codon 5 in exon 1.

BACKGROUND:Previous studies have shown that both intronic and exonic variants in RHD, the gene encoding the D antigen in the Rh blood group system, can alter mRNA splicing at the qualitative and/or quantitative level(s), thus resulting in a D variant phenotype. Here, we sought to further document this mechanism by analyzing extensively RHD variants found in the Chinese population. STUDY DESIGN AND METHODS:From an in-house database of whole exome sequencing of over 27,000 gDNA samples from Chinese individuals, as well as from ten Chinese individuals presenting with a D variant phenotype, RHD variants were selected in a first round of in silico approaches previously used (i.e., Alamut suite splicing module and ΔtESRseq score) for functional analysis by minigene splicing assay. All variants were further reevaluated subsequently by two contemporary bioinformatics tools, that is, SpliceAI and SPiP. RESULTS:From the 269 single nucleotide variants (SNVs) included in the study, the 34 top-score SNVs were selected as potential candidates for altering RHD splicing and comprehensively analyzed at the functional level. We demonstrated that eight SNVs, including three located within the consensus splice sites (c.336-1G>C, c.801+2T>G, and c.940-2delA), four in proximal intronic regulatory regions (c.149-6G>A, c.939+5G>T, c.1154-8T>G, and c.1227+5G>C), and one in exon (c.318G>A) alter splicing with a moderate to severe effect. DISCUSSION:Our findings suggest that combining computational prediction with SpliceAI and/or SPiP using specific settings is an efficient approach (accuracy = 0.941) for selecting candidate SNVs that are prone to disrupt RHD splicing and alter D phenotype subsequently.

BACKGROUND AND OBJECTIVES:Among the rare serologically D-negative (D-) individuals in Asia, those carrying the Asian-type DEL allele (RHD*DEL1) can be safely managed as D+ individuals during transfusion and pregnancy. Recently, some individuals carrying RHD*DEL1, who exhibit serologically weak/partial D phenotypes rather than the serologically D- phenotype, have also been described. Whether anti-D alloimmunization can occur among them was explored. MATERIALS AND METHODS:A retrospective study was carried out in 143 Chinese pregnant women identified as serologically weak/partial D phenotypes. The RHD*DEL1 allele was detected using the high-resolution melting method. Then, RHD genotyping was determined mainly by Sanger sequencing. D epitope expression was detected with the anti-D panel (D-Screen) by haemagglutination and adsorption/elution tests. RESULTS:RHD*DEL1 allele carriers were identified in 42.0% (60/143) of weak/partial D women. The single genotypes (mainly RHD*DEL1/01N.01 or RHD*DEL1/DEL1, n = 52) and the compound heterozygous genotypes (RHD*DEL1/weak or partial D allele, n = 8) were detected. A complete repertoire of D epitopes was shown in six weak/partial D women who simultaneously carried the RHD*DEL1 allele. Alloanti-D was not observed among any carriers (0/60). In the remaining 78 weak/partial D samples available but not carrying RHD*DEL1, 24 types of RHD variant alleles, including six novel alleles, were detected. CONCLUSION:The RHD*DEL1 allele occurred often in the Chinese individuals with weak/partial D phenotypes who showed a lack of anti-D alloimmunization. Routine Asian-type DEL genotyping is recommended both in serologically D- and weak D/partial D individuals with East and Southeast Asian ancestry to consider Asian-type DEL carriers as D+ individuals during transfusion and pregnancy.

Objective To investigate the distribution,phenotype and genotype of D-elute type(Del)in blood donors with RhD negative blood in Guangzhou,so as to understand the molecular biological background of DEL blood group in this area.Methods During the period from November 1,2021 to June 30,2022,the RhD-negative blood initially screened by saline method was confirmed by indirect anti-human globulin test(IAT)serology,and RhCE phenotype was determined by RhCE typing card.A total of 1 146 RhD-negative samples,including all RhD-negative samples with RhCE C+(n=459)and a randomly selected subset of RhCE C-(n=175),were subjected to adsorption-elution(Del)screening(a total of 634 sam-ples).DNA from Del-positive samples was extracted for real-time fluorescent PCR detection of the RHD gene c.1227 locus using high-resolution melting curve analysis(HRM).For samples without mutations detected at the RHD∗1227 locus by HRM,restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP)was performed to amplify the prod-uct which was subsequently digested with Pst I enzyme and analyzed by electrophoresis to determine RHD gene haplotypes.Sanger sequencing was performed for exon sequencing(exon 1-10)of the RHD gene,and gene mutations were analyzed u-sing SeqMan software.Suspected Del-positive samples were subjected to RHD whole gene analysis using third-generation sin-gle-molecule sequencing technology.Results Among the 634 confirmed RhD-negative samples,229(36.1%)displayed Del phenotype,accounting for20%(229/1 146)of the total confirmed RhD-negative samples.The RhCE phenotypes of the 229 DEL cases were as follows:Ccee in 181 cases,CCee in 40 cases,CcEe in 7 cases,and ccEe in 1 case.HRM combined with RHD haplotype analysis showed that there were 170 cases with RHD gene as RHD∗1227A/01N.01,32 cases with RHD gene as RHD∗1227A/1227G,26 cases with RHD gene as RHD∗1227A/1227A,and 6 cases with RHD gene as RHD∗1227G/1227G(sequencing results included 1 case of weak D type 12,4 cases of D-,and 1 case of RHD∗01EL.02).Con-clusion The individual genotype of DEL blood donors in Guangzhou area is mainly characterized by RHD∗1227A/01N.01,and their RhCE phenotypes are all C+.HRM can be used as a molecular biology method for routine screening of Asian-type DEL blood type genes.

TransfusionEarly View ONE-PAGE REPORT OF NEW ALLELES OR ANTIGENS Identification of a novel A4GALT*299A allele associated with the rare p phenotype in one Chinese family Chunyan Mo, Chunyan Mo orcid.org/0009-0003-4943-9524 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorShuangshuang Jia, Shuangshuang Jia orcid.org/0000-0002-0092-8576 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorGuangping Luo, Guangping Luo Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorYanli Ji, Corresponding Author Yanli Ji [email protected] Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China Correspondence Yanli Ji, 31 Luyuan Road, Yuexiu District, Guangzhou, 510095, China. Email: [email protected]Search for more papers by this author Chunyan Mo, Chunyan Mo orcid.org/0009-0003-4943-9524 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorShuangshuang Jia, Shuangshuang Jia orcid.org/0000-0002-0092-8576 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorGuangping Luo, Guangping Luo Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorYanli Ji, Corresponding Author Yanli Ji [email protected] Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China Correspondence Yanli Ji, 31 Luyuan Road, Yuexiu District, Guangzhou, 510095, China. Email: [email protected]Search for more papers by this author First published: 19 March 2024 https://doi.org/10.1111/trf.17795Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. CONFLICT OF INTEREST STATEMENT The authors have disclosed no conflicts of interest. REFERENCES 1Di Ciaccio P, Cutts B, Alahakoon TI, Dennington PM, Soo LA, Curnow J. Clinical consequences of the extremely rare anti-PP1Pk isoantibodies in pregnancy: a case series and review of the literature. Vox Sang. 2020; 116: 591–600. 10.1111/vox.13042 PubMedGoogle Scholar 2Westman JS, Stenfelt L, Vidovic K, Moller M, Hellberg A, Kjellstrom S, et al. Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT. Blood. 2018; 131: 1611–1616. 10.1182/blood-2017-08-803080 CASPubMedWeb of Science®Google Scholar 3Hellberg A, S-MA, Reid ME, Olsson ML. Expression of a novel missense mutation found in the A4GALT gene of Amish individuals with the pphenotype. Transfusion 2007; 48: 479–487. 10.1111/j.1537-2995.2007.01552.x PubMedGoogle Scholar 4Daniels G. Human blood groups. 3rd ed. Hoboken, NJ: Wiley-Blackwell; 2013. 10.1002/9781118493595 Google Scholar 5Wei L, Ji YL, Luo H, Mo CY, Zhang RQ, Zhao Y, et al. Serological and genetic study of a pedigree featuring a rare p phenotype [article in Chinese]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2012; 29: 701–704. CASPubMedGoogle Scholar Early ViewOnline Version of Record before inclusion in an issue ReferencesRelatedInformation

Objective To identify the specificity of alloantibody against high-frequency antigens in one case suffering with severe hemolytic diseases of the fetus and newborn(HDFN)and to screen for matching blood for transfusion.Methods The HDFN test and the antibody serological identification tests in the mother were performed.Several common high fre-quency antigens of maternal red blood cells(RBCs)were determined.IgG subtype coated on the RBCs of the newborn was determined.The phagocytic efficiency of the antibody was tested using the monocyte phagocytosis of sensitized erythrocyte by flow cytometry in vitro.Sanger sequencing of DI gene was performed in the mother,father and mother's brother.The diluted maternal plasma was used for large scale screening of matching blood using IAT in Coomb's gel card.Results Di(b-)phenotype was identified in the mother of the newborn and anti-Dib(titer:512)related HDN was detected in the newborn.IgGl and IgG2 subtypes of anti-Dib were detected and the rate of monocyte phagocytosis was 88.83％(74.7/84.09).The compatible blood was not detected in the maternal relatives.Subsequently,the newborn received the matching RBCs of two Di(b-)donors identified from 5 520 blood donors and discharged from the hospital.We screened out 17 Di(b-)donors out of 51 334 blood donors,indicating that the distribution frequency of Di(b-)among blood donors in Guangzhou was about 0.033％(17/51 334).Conclusion By serology and molecular biology methods,the newborn was identified with HDFN caused by anti-Dib,and an effective large-scale screening method for Di(b-)rare blood types was established to find matching blood,which supported the establishment of rare Di(b-)blood database.

Objective To study the effect of RHAG variants identified in Chinese population on mRNA splicing by minigene splicing assay(MSA) in vitro. Methods The pSplicePOLR2G minigene expression plasmids were constructed for 10 RHAG mutations with relatively high distribution frequency in Chinese population near splicing sites or synonymous mutations by analyzing the RHAG gene data in the KMxD database. Then, the wild-type and mutant plasmids were transfected into HEK 293T cells, and RNA was extracted 48 hours after transfection. After reverse transcription, specific primers were used for PCR amplification, and then agarose gel electrophoresis and capillary electrophoresis were performed to determine whether the mutations will affect the normal splicing of exons. Results MSA in vitro showed that 2 mutations (c.158-5delT, c. 807+ 3A>C) near the splicing site reduced the amount of normal transcripts slightly. The remaining 8 synonymous mutations(c.312G>A, c. 341+ 3G>A, c. 609C>T, c. 681G>A, c. 861G>A, c. 957T>A, c. 984T>C and c. 1139-7G>A) had no impact on the splicing of RHAG mRNA. Conclusion This study showed that RHAG gene was conservative in terms of splicing, and the mutations near splicing sites and synonymous mutations were less likely to cause abnormal splicing of RHAG gene.

Red blood cells (RBCs) of the Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) in routine testing. RhD-positive (D+) RBC transfusion for Asian-type DEL patients has been proposed but has not been generally adopted due to a lack of direct evidence regarding its safety and underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in Asian-type DEL patients; none of the recipients (0/42; 95% confidence interval, 0%-8.40%) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4,045 serologic D- pregnant women throughout China; alloanti-D was found only in true D- individuals (2.63%, 79/3,009), but not in those with Asian-type DEL (0/1,032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs following transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in Asian-type DEL patients, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as NCT03727230.

Objective To establish a high-throughput detection method for ABCG2*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. Methods The specific primers were designed and synthesized, the sample carrying homozygous ABCG2*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2*376T alleles were sequenced to confirm the accuracy of the HRM method. Results A HRM method, which can detect ABCG2*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. Conclusion The HRM method can be used to accurately screen and type ABCG2*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

Objective To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. Methods ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. Results The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. Conclusion In this case, the development of anti-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.