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Objective To analyze the correlation between the expression of soluble glycoprotein A(GPA)in plasma of healthy blood donors and anti-M and anti-"Mia"antibodies.Methods Plasma from healthy donors from February 9,2022 to February 15,2023 was collected:irregular antibody-negative NN type(group Ⅰ,n=118)and MM type(group Ⅱ,n=51),anti-M antibody positive NN type(group Ⅲ,n=145)and anti-"Mia"antibody positive companion type(group Ⅳ,n= 87),the GPA content in plasma of different individuals in 4 groups was detected,and the difference in GPA expression was analyzed by t-test.Results The average plasma GPA contents in groupsⅠ,Ⅱ,Ⅲ and Ⅳ were 9.941±0.252,10.97±0.256,5.139±0.129 and 4.28±0.139ng/ml,respectively.The average GPA content of groups Ⅰ and Ⅱ was higher,and the average GPA content of groups Ⅲ and Ⅳ was lower,and the differences were statistically significant(all P<0.01).Conclusion The GPA content in plasma of healthy donors with anti-M and anti-"Mia"antibodies was significantly lower than that of the antibody-negative group.The results of this study lay a foundation for further investigation of whether GPA in plasma has the ability to neutralize anti-M and anti-"Mia"antibodies,improve disease diagnosis and safe blood transfusion.
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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.
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Humans , Blood Group Antigens/genetics , Genotype , Genotyping Techniques , Heterozygote , Alleles , Blood Donors , Rh-Hr Blood-Group System/geneticsABSTRACT
【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.
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【Objective】 To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. 【Methods】 Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. 【Results】 Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. 【Conclusion】 The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.
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【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.
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【Objective】 To analyze the blood samples sent by hospitals in Shenzhen to solve ABO cross-match incompatibility during 2011 to 2020, so as to find corresponding solutions to improve the efficacy of blood transfusion. 【Methods】 The clinical data of 1 770 cases of cross-match incompatibility in our laboratory from January 2011 to December 2020 were collected and reviewed. The causes of cross-match incompatibility were analyzed, the types of unexpected antibodies were determined. The overall incidence of antibodies was evaluated by statistical method of classified variables. The safety of blood transfusion was safeguarded by ABO homotype plus cross-matching compatibility. 【Results】 1) The 1 770 samples, presenting cross-matching incompatibility, involved 956 patients. The average number of cross-matching per patient from 2011 to 2015 was 1.32(307/232), which increased from 1.27(103/81) in 2016 to 2.23(286/128) in 2018, and remained stable in 2019 and 2020. 2) Among 956 patients, auto-and/or allo-antibody in plasma were yielded in 90.38%(864/956), including auto-antibody plus alloantibody in 42.26%(404/956), solo auto-antibody in 20.71%(198/956) and solo allo-antibody in 27.41%(262/956). Up to 20 kinds of specific allo-antibodies were detected, belonging to 8 blood groups. Among them, 70.82%(551/778) were Rh blood group, such as anti-E(37.15%)>anti-c(20.95%)>anti-C(5.27%)=anti-e(5.27%)>anti-D(2.19%), followed by 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)] and H [0.26%(2/778)]. 3) 86%(37/43) of multiple transfusion recipients, aged below 20 years old, were thalassemia, and 1-4 kinds of allo- and/or auto-antibody were yielded. 【Conclusion】 The cross-matching incompatibility were mainly caused by allo- and/or auto-antibodies, which may be induced by blood transfusion, pregnancy or autoimmune diseases such as autoimmune hemolytic anemia.Those suspicious blood samples in clinical should be sent to blood group reference laboratory for further determination, in order to ensure the safety and efficacy of blood transfusion.
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【Objective】 To analyze the polymorphism of erythrocyte membrane blood group glycoprotein A (GPA) related gene GYPA in high and low endemic population for clonidia sinensis infection, aimed at investigating the correlation between erythrocyte transmembrane glycoproteins and clonorchis sinensis infection. 【Methods】 From Dec 2019 to Jun 2020, anticoagulant blood samples were randomly collected in WuMing district (n=700) and GuiGang district (n=500 ) of Nanning city, and the IgG antibody to clonorchis sinensis in plasma was detected, and the DNA of leukocyte was extracted. The full-length exon and partial intron of GYPA gene were sequenced, mutations were characterized by gene cloning, and the risk of infection was calculated by chi-square test. 【Results】 The yield rate of IgG antibody was 62.7% (439/700) in WuMing district and 3.4% (17/500) in GuiGang district(P<0.05). The insertion of base C at the 54th position of intron-2 in GYPA gene caused the reading frame shift. The mutation was presented in 23.9% (105/439) and 17.6% (3/17) of the population with clonorchis sinensis exposure in WuMing and GuiGang area, respectively, while 49.4% (129/261) and 54.7% (264/483) in the negative population, respectively (P<0.05). 【Conclusion】 The infection rate of clonorchis sinensis in WuMing district was higher than that in GuiGang district. The mutation rate of reading frame shift caused by the insertion of base C at the 54th position of GYPA intron-2 was much lower in the positive population of clonorchis sinensis infection than the negative population, suggesting that the mutation is a protective gene in the negative population of clonorchis sinensis infection. It is necessary to study the mechanism of clonorchis sinensis infection and the mutation point of this gene in order to facilitate the early diagnosis of disease, blood transfusion management, treatment and prevention.
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Objective To study the molecular genetic background of Bel subtype at ABO blood group.Methods Three samples and fifteen samples were diagnosed as Bel subgroup and normal control samples by serological test,respectively.The extracted DNA was genotyped by sequence specific primer- polymerase chain reaction foilowed by sequencing for Exon6 and exon7 at ABO locus and clones were sequenced.Results A novel Bel variant allele(GenBank EF117687) was identified in a Bel individual.The Bel allele was different from the regular B101 allele by single 952G>A missense mutation in exon7.resulting in an amino acid subsfitution of Val for Met at 318 locus.No mutations were detected in the fifteen control samples and the other two Bel allele samples.Conclusions The mutation position was fimt found to lie on coding region of ABO gene behind nucleotide 930.The mutation of G952A in the al,3 galactosyhransferase gene may be one of the molecular genetic basis of Bel ohenotype.
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Objective To investigate serological blood typing of the ABO locus which contradict to general law of inheritance in parentage,and the underlying reasons for severe hemolytic disease of newborn(HDN).Methods To research the family whose newborn is AB phenotypes,mother is O phenotypes and father is AB phenotypes.The familiy were genotyped by parentage tests, serological tests,PCR-SSP and direct DNA sequencing at exons 6 and 7 of ABO gene.At the salne time,HDN was detected by micro column gel Coombs (MGCT), and the primary fingerposts of the routine blood tests. Biochemical tests were dynamically observed.Results The results of parentage tests showed that three-generation pedigree have parent-child relationship. The red blood cell(RBC)of this AB phenotypes of this family members strongly agglutinated(4+)with diverse monoelonal anti-A and anti-B antibodies,and their serum did not contain anti-A and anti-B antibodies in blood anti-typing.PCR-SSP can not detect their A and B gene,but DNA sequencing at exons6 and 7 of ABO gene revealed that it had the B(A)803C→G mutation.Conclusions The genetm basis of this parentage are B(A)803G blood gene which harbored both A and B difunctionality of glyeosyhransferases.This was the first report that severe HDN resulting from a large number of A and B antigens in RBC of B(A)phenotype of a newborn,which has clinical significance on ABO locus.
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Objective To develop an efficient method for detecting the short tandem repeat(STR) of fetal DNA in maternal plasma by miniSTR technique.Methods A total of 9 blood samples form pregnant women from 11 to 27 weeks of gestation were collected.Each isolated total plasma DNA was amplified in single multiplex using the ABI MiniFilerTM kit,which could simultaneously genotype the 9 miniSTR loci,including D13S317,D7S820,D2S1338,D21S11,D16S539,D18S51,CSFIPO,FGA and Amelogenin,and the PCR products were detected by using ABI PrismTM 3100 DNA Sequencer.The allelic designation of each STR locus was accomplished using the GeneMapper ID 3.2 software.Results Father-origin fetal STR allele was detected in all the 9 plasma DNA samples.An average of 3.1 fetal STR alleles of the 8 autosomal STR loci was observed in each of the 9 plasma DNA samples.As for the Amelogenin locus,Amelogenin Y allele was detected in 5 plasma DNA samples from pregnancies with male fetus,and allelic peak height values were all over 50 RFU,according to ABI Mini FilerTM PCR conditions,and the ratio of Amelogenin Y allele peak height value to Amelogenin X allele peak height value was 8.45%.However,no Amelogenin Y allele was detected in other 4 plasma DNA samples from pregnant women with female fetus.Conclusion The miniSTR technique is suitable for STR genotyping using fetal DNA in maternal plasma,and it suggests a broad application in noninvasive molecular prenatal diagnosis.
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<p><b>OBJECTIVE</b>To study the molecular genetic background of B subtype in Chinese Han population and identify novel allele at the ABO locus.</p><p><b>METHODS</b>Ten samples from randomly selected blood donors of normal B phenotype used as control, and six samples from individuals diagnosed as B subgroup by serological tests were genotyped by sequence specific primer polymerase chain reaction and direct DNA sequencing at the exons 6 and 7 of ABO gene. The exons 6 and 7 and the intervening intron 6 of the B allele from each B subgroup sample were analyzed by cloning and haplotype sequencing.</p><p><b>RESULTS</b>A novel B variant allele was identified in two individuals whose blood samples were diagnosed as belonging to Bx subgroup and Bw subgroup respectively, the novel B allele being different from the allele B101 by single 695T>C missense mutation in exon 7. A family with the individual possessed Bx subgroup was studied. Among 22 family members tested, seven family members were found to carry the novel B variant allele. No novel point mutation at the exons 6 and 7 of ABO gene were detected in the ten control samples and the other four samples with B subgroup.</p><p><b>CONCLUSION</b>The present authors define this allele as a novel B variant allele. The mutation of this novel allele, in which the nucleotide alters from T to C at position of 695 in exon 7 and hence results in an amino acid change from Leu to Pro, is expected to diminish the enzyme's activity. It indicates that the alteration of amino acid at the position of 232 is critical to the activity of glycosyltransferases.</p>
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Female , Humans , Male , ABO Blood-Group System , Genetics , Alleles , Asian People , Genetics , China , DNA Mutational Analysis , Exons , Family Health , Introns , Mutation, Missense , Pedigree , Polymerase Chain ReactionABSTRACT
BACKGROUND: ABO is the most important blood group system for blood transfusion. Though widely used in determining ABO blood group for its simplicity and rapidity, serological technology has its inherent limitation, for which ABO genotyping provides a valuable alternative.OBJECTIVE: To study ABO gene polymorphism in Chinese Han population and apply ABO genotyping technique to solve serological problems in clinical practice of blood transfusion.DESIGN: Comparison of ABO genotyping results of random selected samples with those of routine serological phenotyping.SETTING: An institute of transfusion medicine in a municipal blood center.PARTICIPANTS: Totally 260 unrelated healthy Chinese blood donors of Han nationality were randomly selected in Shenzhen Blood Center from March to December in 2002, including 110 male and 150 female subjects aged between 18 and 50 years. A sample with discrepancy in serological ABO phenotype was from our blood center, and the donor' s family was investigated. Six samples suspected to be A2 phenotype by serological test were from four hospitals in Shenzhen including the Second People' s Hospital of Shenzhen.METHODS: The DNA was extracted from the peripheral blood by rapid salt fractionation, and subjected to polymerase chain reaction(PCR) with sequence-specific primers (PCR-SSP) to amplify the ABO gene for ABO genotyping. The alleles of the blood type difficult to determine were amplified with PCR-SSP on the basis of serologic tests including absorption and elution test and agglutination inhibition assay of salivary blood-group substances.MAIN OUTCOME MEASURES: Genotypes and phenotypes of the blood samples from 260 individuals and of the samples with serological ABO discrepancy.RESULTS: In the 260 Chinese Han individuals, in accordance with Hardy-Weinberg equilibrium, the gene frequencies of O1, B, A1O1(A467c), A1O2/1O3(A467T) alleles were 0. 582 7, 0. 184 6, 0. 009 6, and 0. 2231, respectively. Two of the six individuals with difficulty of blood type determination and suspected to have A2 phenotype by serological tests proved to have A2O1O1 genotype, and the rest were all of A1O2/A1O3O1. Three children of a family with difficult identification were para-Bombay types, and their ABO types were A102B, A102B and A102O1, respectively.CONCLUSION: ABO PCR-SSP genotyping is simple, rapid and accurate and can be a valuable complement to serological identification.
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Objective To study the mutation of FUT1 and FUT2 genes in para-Bombay individual.Methods Direct DNA sequencing of FUT1 and FUT2 gene coding region were analyzed in two individuals with para-Bombay phenotype.Results One individual lost one of the three AG repeats located at nucleotides 547~552 of the FUT1 gene, whereas two of the three T repeats located at nucleotides 880~882 were deleted in the other.Conclusion Two frame-shift mutations of FUT1 gene are responsible for the H antigen deficiency
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T missense mutation in exon 7. No novel point mutation at exons 6 and 7 of ABO gene was detected in the other four samples with B subgroup. Conclusion We define this allele as a novel B allele in Chinese Han individuals. The mutation of this novel allele in which the nucleotide changes from C to T at position 721 in exon 7, resulting in an amino acid change from Arg to Trp, results in the decrease of the enzyme activity. It indicates that the alteration of amino acid at position of 241 is critical to the activity of glycosyltransferases.