OBJECTIVE:By analyzing the correlation between genotypes and phenotypes, we explored the impact of the variant c.917T>C (p.L306P) in the ABO*B.01 allele on the expression and function of B-glycosyltransferase (GTB). This study aims to elucidate the molecular mechanisms underlying the occurrence of this subtype. METHODS:The study subjects included a blood donor specimen with incompatible forward and reverse ABO typing results. ABO phenotyping was determined using ABO blood group serology and GTB activity testing. Subsequently, Sanger sequencing and third-generation sequencing based on the PacBio platform were employed to sequence the ABO gene, resulting in the determination of haplotype sequences. Mutations were identified through sequence alignment. An in vitro cell expression system was established to assess the impact of the mutation site on antigen expression. RESULTS:The index case in this study was identified as B subtype with the allelic genotype c.917T>C in ABO*B.01/ABO*O.01.01 , which has not been previously reported. in vitro expression results revealed decreased levels of GTB expression and overall GTB activity in the mutant cells. Furthermore, the expression of the B antigen on the cell membrane was weaker in the mutant cells compared to the wild-type cells. CONCLUSION:The p.L306P variation caused by the c.917T>C mutation in the ABO*B.01 allele may be a genetic factor contributing to the reduced expression of B antigens on the surface of red blood cells.

By analyzing the correlation between genotypes and phenotypes, we explored the impact of the variant c.917T>C (p.L306P) in the ABO*B.01 allele on the expression and function of B-glycosyltransferase (GTB). This study aims to elucidate the molecular mechanisms underlying the occurrence of this subtype. The study subjects included a blood donor specimen with incompatible forward and reverse ABO typing results. ABO phenotyping was determined using ABO blood group serology and GTB activity testing. Subsequently, Sanger sequencing and third-generation sequencing based on the PacBio platform were employed to sequence the ABO gene, resulting in the determination of haplotype sequences. Mutations were identified through sequence alignment. An in vitro cell expression system was established to assess the impact of the mutation site on antigen expression. The index case in this study was identified as B subtype with the allelic genotype c.917T>C in ABO*B.01/ABO*O.01.01 , which has not been previously reported. in vitro expression results revealed decreased levels of GTB expression and overall GTB activity in the mutant cells. Furthermore, the expression of the B antigen on the cell membrane was weaker in the mutant cells compared to the wild-type cells. The p.L306P variation caused by the c.917T>C mutation in the ABO*B.01 allele may be a genetic factor contributing to the reduced expression of B antigens on the surface of red blood cells.

Background: RhD variants are categorized into partial D, weak D, and DEL. The detection of DEL can only be achieved through the adsorption and elution method or molecular techniques. Here, we report a case of DEL phenotypes associated with a novel allele in a Chinese individual. Study design and methods: We used serological methods such as saline, indirect anti-human globulin, and adsorption-elution. The RHD genotype was determined by the PCR-sequence specific primer (PCR-SSP) method as well as the Sanger dideoxy sequencing. Results: RBCs of the sample were found to be DEL phenotype by serological testing, with negative reactions in the saline and indirect anti-human globulin tests while positive reactions by the absorption-elution method. The genotyping results revealed a hemizygous ( RHD c .1127 T>G / RHD-). The novel allele sequence has been submitted to GenBank (Accession number: OR608456). Conclusion: Our study demonstrates a case of a Chinese individual with DEL phenotype caused by a novel allele RHD c .1127 T > G. It expands the database of the DEL variant.

OBJECTIVE:To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-).METHODS:Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors.RESULTS:The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰.CONCLUSION:A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.

Several cases of the hemolytic disease of the fetus and newborn (HDFN) caused by immunoglobulin G (IgG) anti-M antibodies have been reported, in which almost all the HDFN-associated anti-M were warmly reacting. Here we report two cases of severe HDFN associated with cold-reacting IgG anti-M. In both cases, pregnancy was terminated, in weeks 33 and 23 respectively, due to a diagnosis of fetal growth retardation (FGR). To our knowledge, these are the most severe HDFN cases caused by cold-reacting IgG anti-M.

OBJECTIVE:The characteristics of the full-length mRNA sequences of MNS blood group-related genes GYPA, GYPB and GYPE were analyzed to understand the polymorphism of MNS blood group genes.METHODS:Anticoagulated blood within 24 h from 500 unpaid blood donors (8 ml each) were randomly selected, and MN, Ss and Mia blood types were identified by serological methods. 5 samples with different combinations of MNS and Mia blood types were randomly selected from 500 samples, and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, then total mRNA was extracted. cDNA was prepared by using the reverse transcription kit. The target fragments were amplified by nested PCR, and the full-length mRNA sequences of GYPA, GYPB and GYPE were sequenced after gel cutting and recycling, and the base sequences were analyzed by Oligo 6.0 software.RESULTS:The MN, Ss and Mia phenotypes were detected by serological methods, and there were differences in agglutination intensity of red blood cells (RBC) and anti-Mia serum between different individuals. The full-length mRNA sequences of GYPA, GYPB and GYPE genes in 5 samples of different phenotype combinations were detected. The exon-6 was completely deleted from the GYPA mRNA in 1 sample, and the full-length of GYPA mRNA in the other 4 samples were complete. The exon-2 was completely deleted from the GYPB mRNA in 2 samples, with Mia blood type negative. 2 samples showed complete full-length of GYPB mRNA, with Mia blood type positive. There was base substitution in exon-5 of GYPB mRNA in 1 sample. The full-length of GYPE mRNA was intact in 5 samples.CONCLUSION:MNS blood group related-genes have obvious polymorphism, and the detection of full-length mRNA sequence lays a foundation for the analysis of GYPA, GYPB and GYPE gene structure and in-depth study of MNS blood group antigen expression.

Objective To investigate the family inheritance of α-Thalassemla gene and the risk of severe anemia in neonates caused by cold IgG anti-M. Methods ABO, Rh, MN blood groups and the specificity of unexpected antibody were identified by blood group serology. The IgG subtype and antibody titer of anti-M antibody were detected. The etiology of neonatal hemolytic disease was identified by three tests and α-Thalassemla gene diagnosis. Results Family investigation showed that father was B, CCDee, MN with no α-Thalassemla gene detected; Mother B, CcDee, NN, carrying α-Thalassemla gene; both the proband and his brother were B, CCDee, MN, carrying α-Thalassemla gene. Cold IgG anti-M was present in plasma of both the mother and the proband. The titer of the mother was 128 and that of the proband was 64. The subtype of IgG anti-M was IgG1 and IgG3. The direct anti-globulin test, release test and free test of the proband and his brother were negative, and the diagnosis was severe anemia and hemolysis caused by α-Thalassemla combined with cold IgG anti-M. Conclusion The direct antiglobulin test of neonatal hemolytic disease caused by IgG anti-M can be negative or weakly positive, and α-Thalassemla gene could be hereditary in families. The presence of α-Thalassemla gene can cause anemia, hemolysis and splenomegalysis in neonates, which could be aggravated when accompanied by cold-type IgG anti-M. In the presence of high-valency IgG antibody in plasma, blood exchange combined with transfusion can improve the curative effect.

Objective To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure. Methods The 9 blood samples with rare RhD variants were collected from 210 644 blood donors of Shenzhen Blood Center. Regular serological assaying was used for determination of Rh type for the 9 samples. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and to screen the antibodies. D-screen reagent was sued to analyze the RhD epitopes of the samples. RHD zygosity testing of the samples was detected by PCR-SSP. The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced. The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN, SIFT, PolyPhen-2 and MutationTaster software. Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD. Results A total of 9 individuals with rare RhD variants were identified as follows: RHD*weak D type 25, RHD*weak D type 50, RHD*weak D type 95, RHD*weak D type 12, RHD*weak D type 128 and four novel RHD alleles. The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples. Conclusion Five rare and four novel RHD alleles have been identified. Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles. Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.

OBJECTIVE:To establish a based method flow cytometry to identify the antigen Jka in human red blood cells (RBCs) and verify its accuracy.METHODS:A total of 96 blood samples were enrolled in the study randomly from the voluntary blood donors in Shenzhen Blood Center. The RBCs were incubated with IgG anti-Jka primary antibody, and then labeled with the secondary antibody anti-IgG-Alexa Fluor 647. The fluorescence histograms of each sample were obtained by flow cytometry. Serological agglutination test was used to compare the accuracy of flow cytometry in the detecting of antigen Jka, while PCR-SSP and gene sequencing genotyping were used to verify the accuracy of flow cytometry in the detecting of the antigen in human RBCs.RESULTS:The results of flow cytometry for antigen Jka in human RBCs were consistent with those from serological tests. Samples that demonstrated higher serological agglutination intensity also showed higher fluorescence activity, which indicate more stronger of Jka antigen. The sensitivity of flow cytometry was higher than that of serological test; especially in distinguish Jka weak and negative samples. Flow cytometric results of all samples were consistent with the genotyping results, which confirmed the accuracy of flow cytometry.CONCLUSION:The study established a new flow cytometry-based method successfully for the identification of Jka antigen of Kidd blood group in human RBCs. The Kidd blood group antigen Jka of different intensities can be accurately distinguished by the technique.

BACKGROUND:The null phenotype in P1PK blood group, known as "p," is extremely rare in the whole world. Individuals of p phenotype spontaneously form anti-PP1PK isoantibody. Here, we report a case of p phenotype with naturally occurring anti-PP1PK isoantibodies in a Chinese individual.STUDY DESIGN AND METHODS:Serology tests, containing alloantibodies screening and identification, were conducted to demonstrate the phenotype in P1PK blood group. The genotype of A4GALT gene was identified by haplotypes separation and sequencing.RESULTS:The serological assay demonstrated the p phenotype of the proband, presenting with 1:64 titer of anti-PP1PK . The sequencing data revealed a compound heterozygote consisting of A4GALT*P1.01 with c.343A>T and a novel allele based on A4GALT*01N.05 with an addition polymorphism c.100G>A. The sequence of the novel allele has been submitted to GenBank and the accession number OM912503 was assigned.CONCLUSION:Our study demonstrates a case of naturally occurring anti-PP1Pk in a Chinese individual with p phenotype, which is based on compound heterozygosity including one novel allele. As the proband is a young lady, monitoring the titer of anti-PP1PK and early initiation of medical intervention are essential after her pregnancy.

目的 探讨α-Thalassemla基因的家系遗传及合并冷型IgG抗-M导致新生儿重度贫血的危害.方法 以血型血清学方法鉴定家系ABO、Rh、MN血型与意外抗体特异性,检测抗-M IgG亚型及抗体效价,结合新生儿溶血病诊断的三项试验与α-Thalassemla基因的诊断,明确病因后换血与输血治疗.结果 家系调查显示父亲:B,CCDee,MN,未检到 a-Thalassemla 基因;母亲:B,CcDee,NN,携带 α-Thalassemla 基因;先证者与弟弟均为:B,CCDee,MN,携带 α-Thalas-semla基因.母亲与先证者的血浆均存在冷型IgG抗-M,母亲抗体效价:128,先证者抗体效价:64.IgG抗-M亚型为IgG1与IgG3型,先证者与弟弟的直接抗球蛋白试验阴性,放散试验阴性,游离试验阳性,诊断为α-Thalassemla合并冷型IgG抗-M引起重度贫血与溶血.结论 由IgG抗-M引起新生儿溶血病的直接抗球蛋白试验可以表现为阴性或者弱阳性,α-Thalassemla基因存在家系遗传.携带α-Thalassemla基因可以引起新生儿贫血、溶血和脾肿大,合并冷型IgG抗-M可加重胎儿期及新生儿期的贫血与溶血程度,血浆中存在高效价IgG抗体时,换血合并输血可以提高疗效.

目的 研究1例RhD血型鉴定部分凝集结果个体及其家系血清学表现和RHD基因.方法 通过血型微柱凝胶卡检测先证者ABO及RhD血型;盐水试管法检测先证者及其父母RhCcEe抗原;间接抗人球蛋白试验(indirect anti-human globulin test,IAT)及流式细胞术检测先证者RhD抗原.PCR序列特异性引物(PCR sequence specific primer,PCR-SSP)检测RHD基因以及RhD杂合型分析,基因测序方法分析RHD基因编码区序列.结果 血清学检测发现先证者血型为A型RhCcee,血型微柱凝胶卡、盐水试管法以及IAT法检测RhD抗原,结果呈部分凝集现象.流式细胞术结果显示先证者RhD抗原性减弱.经RHD基因编码序列分析发现,RHD基因第9外显子上的第1212位碱基发生C>A纯合突变,为RHD*weak D type 72的特征性突变点.家系调查显示,先证者父亲为O型RhCCDee,母亲为A型RhCcDee.父亲携带RHD*weak D type 72等位基因,基因型为RHD*weak D type 72/RHD+;母亲一条染色体缺失了全部的RHD基因,基因型为RHD+/RHD-.证明先证者分别从父亲和母亲遗传RHD*weak D type 72和RHD-等位基因,基因型为RHD*weak D type 72/RHD-.结论 发现了1例RHD*weak D type 72/RHD-基因型个体,丰富了RHD*weak D type 72变异型的研究数据.根据家系调查证明,RHD*weak D type 72等位基因由遗传获得,而非由个体基因变异形成.

The authors have disclosed no conflicts of interest. Appendix S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND AND OBJECTIVES:The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system.MATERIALS AND METHODS:Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins.RESULTS:Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins.CONCLUSIONS:Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

目的 研究深圳地区血清学弱D表型献血者RhCcEe表型与RHD基因型特点.方法 收集38例献血者血液样本,抗-D抗体(IgM/IgG)检测RhD抗原.采用IgM抗-C、抗-c、抗-E、抗-e抗体检测RhCcEe抗原.利用PCR-SSP方法分析RHD基因10个外显子,必要时对RHD基因的所有外显子进行直接测序.统计学方法分析RhCcEe表型与RHD基因型之间的相关性.结果 38例血清学弱D表型献血者中发现8种已报道的等位基因,RHD*weak partial 15(15/38)、RHD*DEL1(11/38)、RHD*DVI.3(5/38)、RHD*weak D type 61(1/38)、RHD*weak D type 95(1/38)、RHD*DCC(1/38)、RHD*DFR1(1/38)、RHD*weak D type 50(1/38)、RHD*weak partial 15/RHD*DEL1杂合(1/38).其中RHD*weak D type 50在中国汉族人群中首次报道.另外,发现了1例新的RHD等位基因RHD*IVS9-1C(c.1228-1G>C).该等位基因的序列数据已提交GenBank,登记号为MT755965.经相关性分析,RHD*weak partial 15与RhE抗原相关系数为0.727(P<0.01),RHD*DEL1与RhC抗原相关系数为0.645(P<0.01).结论 深圳地区血清学弱D表型献血者的RHD基因结构呈现多态性,主要为RHD*weak partial 15、RHD*DEL1、RHD*DVI.3,并存在其他稀有的基因型;RHD*weak partial 15与R h E抗原、RHD*DEL1与RhC抗原存在显著正相关.

The polymorphism of MNS blood group antigens is determined by the amino acid sequences of glycophorin A (GPA) and glycophorin B (GPB). Genetic mutations in their coding genes GYPA and GYPB might lead to the changes of amino acid and influence the expression of antigens. The determinants of the polymorphism of M and/or N antigens are the 1st and 5th amino acids of mature GPA protein (residues 20 and 24 counting from the translation-initiating site), while the polymorphism of S and/or s antigens are determined by the 29th amino acids of mature GPB. 2 The allelic frequency of GYPA and GYPB demonstrates regional differences in human genetics 3 We are committed to exploring the genetic polymorphism of these genes. Here, we describe a novel mutation, c.125G > C, on the GYPA*01 allele in a 28-year-old Chinese male, which caused absence of M antigen in red blood cells (RBCs).

The authors have disclosed no conflicts of interest. Fig. S1 Genotype. The top chromatogram (direct sequencing) shows the heterozygous sequence (T/C at nt.917). The lower two chromatograms (after cloning and sequencing) show separate nucleotide sequences from each allele. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Figure S1. Genotype. The top chromatogram (direct sequencing) shows the heterozygous sequence (delA at nt.36–39) of the proband. The lower two chromatograms (after cloning and sequencing) show separate nucleotide sequences from each allele. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

目的 总结并探讨深圳地区最近10年临床疑难交叉配血(标本)不合原因,为提高输血疗效提供解决方案.方法 收集2011年～2020年深圳地区医疗机构送本中心做疑难血型鉴定的1770例临床标本,分析其交叉配血不合的原因,确定患者血浆中意外抗体的类型,通过分类变量的统计学方法对抗体做总体率评估.以同型输血结合相容性相输的原则来提高输血的安全性.结果 1)1770例疑难配血标本来源于956名患者,其中2011年～2015年疑难配血患者人均配血次数为1.32(307/232);自2016年始,疑难配血患者人均配血次数由2016年的1.27(103/81)上升至2018年的2.23(286/128),并在之后2年保持稳定.2)在956名其交叉配血不合患者中,由自身抗体或/和同种抗体阳性的比例为90.38％ (864/956),包括42.26％ (404/956)自身抗体合并同种抗体阳性、20.71％(198/956)单纯自身抗体阳性、27.41％(262/956)单纯同种抗体阳性;检出特异性同种抗体20种,分属于8个血型系统,其中Rh血型的55种抗体的频率达到70.82％(551/778)[抗-E(37.15％)＞抗-c(20.95％)抗-C(5.27％)=抗-e(5.27％)＞抗-D(2.19％);其后依次为MNS[11.40％(112/778)]、Kidd[5.66％(44/778)]、Leiws[3.21％ (25/778)]、Duffy[1.80％(14/778)]、Diego[1.03％ (8/778)]、P1[0.39％ (3/778)]、H[0.26％ (2/778)]血型系统的同种抗体.3)年龄＜20岁的多次输血患者中有86％ (37/43)为地中海贫血患者,均检测到自身抗体或红细胞同种抗体1～4种.结论 临床交叉配血不合的原因主要是由输血或妊娠免疫导致的同种抗体、自身免疫性疾病如自身免疫性溶血性贫血引发的自身抗体干扰配血所致;临床医疗机构如遇交叉配血不合,应立即送至具有检测能力的血型参比实验室鉴定和配血,这对于保障临床安全用血与提高疗效至关重要.

Figure S1. Sequencing results of exon 10 and flanking intron region of RHD. (A) Sequence analysis result of the proband. The arrow points to RHD c.1228-1G>C. (B) Sequence analysis result of the daughter. The arrow points to RHD c.1228-1G/C. Figure S2. Family pedigrees of the proband with a RHD c.1228-1G>C allele. The D haplotype carrying a RHD c.1228-1G>C allele was found in the proband (black background) and also detected in the daughter. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.