B para-Bombay phenotype in China caused by homozygous mutation for site 328 G>A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah^328G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

B para-Bombay phenotype in China caused by homozygous mutation for site 328 G > A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah3^28G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

Serological?identification and molecular?characterization?of?B?(A)?02?subtype?in?patients?and blood donors from?Eastern?China

This study was designed to identify the rare?type?ABO?blood?groups, B(A) 02, from Eastern China. Three samples with discordant serological results during routine blood type identification and four samples from one sample’ family were selected. All of them were detected by serological method. The exon 6 and 7 of the ABO genes were amplified by PCR and sequenced. They were typed as AsubB by serology and as BO by genotype. In AsubB samples, nt 700C>G mutation was detected in B gene, which was previously defined as B(A)02 alleles. In these seven samples, six showed B(A)02/O01 and one showed B(A)02/O02.B(A)02 allele was found to be more common in this study than B(A)04 which is considered to be more frequent than B(A)02. The careful identification of rare blood types is important for the safety of clinical blood transfusion.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h . The serological method was applied to identify the proband''s ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C>T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h. The serological method was applied to identify the proband's ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C > T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

Serological and genetic analysis of a rare CisAB01/O01 blood group

The paper aims to analyze a rare blood sample in Ganzhou City Hospital with CisAB subtype and explore a feasible pattern for blood typing of rare blood type patients, so as to ensure clinical transfusion safety. The routine serological methods were used for ABO forward and reverse blood typing and the fluorescence real-time PCR technique was used for sample genotyping. A human ABO blood group 6-7 exon sequencing kit was used for sequence analysis. The nucleic acid sequence of the sample was compared with reference sequences. The forward typing results demonstrated that the sample was ABw, RhD positive. The sample exhibited 4+ agglutination with anti-H and anti-AB antibodies. Reverse typing by microcolumn gel method showed an AB result, but the serum sample demonstrated weak agglutination with B cell under room temperature, 4 °C and 37 °C in saline when tested with tube method respectively. The serological results matched with the A₂B₃ serotype. The fluorescent real-time PCR genotyping results displayed A/O01. The sequence analysis demonstrated deletion of guanine in 261-position 467C>T (heterozygote) and 803G>T (heterozygote) mutation respectively. The mutation caused the A glycosyltransferase peptide chain to change from proline to leucine (P156L) at 156 and from glutamate to alanine (G268A) at 268. The result demonstrated that the sample''s genotype was CisAB01/O01. The mutation of glycosyltransferase coding gene leads to an abnormal serological reaction pattern. Only by combining the results of genetic analysis can we get the true sample blood type and better ensure the safety of clinical blood transfusion.     

Rare B(A)02/O01 was found in Sichuan again: a case report

B(A) is a rare ABO blood subgroup. Here we reported a B(A)02/O01 case. One 25-year-old female patient showed inconsistent forward and reverse blood grouping results based on micro-column gel agglutination assay. PCR-SSP and PCR-SBT based genotyping indicated that the patient was B(A)02/O01 heterozygous.     

Serology and gene sequence analysis of rare subgroup A3 blood group

To investigate the serological phenotypic characteristics and possible mechanism of subgroup A₃, a blood donor's ABO phenotypes were detected by the conventional microcolumn gel method and classic tube method. N-acetylgalactosaminyl transferase activity was detected by the non-radioactive phosphate coupling method. ABO subtype genotyping was determined by PCR-SSP and exons 1-7 of ABO gene were analyzed by Sanger sequencing. The donor's blood type was subgroup A₃ as evaluated by serological test. There was no N-acetylgalactosaminyl transferase activity in the red blood cells and weak N-acetylgalactosaminyl transferase activity in the plasma. The ABO blood group genotyping result was ABO*AO1, and the gene sequencing result was confirmed as A221/O01. Sequencing results showed two mutations, 467C>T and 607G>A in exon 7 in ABO*A allele. In conclusion, it is suggested that the ABO blood group of the donor be subgroup A₃, which may be induced by mutations 467C>T and 607G>A, and led to a decrease in N-acetylgalactosaminyl transferase activity and resulted in weakened A antigen.     

ABO genotyping by PCR-RFLP and cloning and sequencing

A refined PCR-RFLP based method was established to genotype ABO blood groups. The main objective of this study was to make the techniques also suitable for working with degraded DNA. Specific primer design was carried out to choose fragments shorter than 200 bp as necessary in forensic and archaeological applications. Four fragments of exon 6 and 7 of the ABO gene were amplified and digested by in total 7 restriction endonucleases. Particular attention was paid to the base changes at nucleotide positions 261(delG), 297, 526, 703, 721, 771, 796 and 1060(delC) in order to distinguish the six common alleles A101, A201, B, O01, O02 and O03. Furthermore, this method also enables determination of seven of the less frequent alleles: A104, A204, Ax02, Ax03, O05, O06 and O07. The method was applied successfully to a series of blood samples with known phenotypes and genotypes as well as DNA extracted from a thirty year old blood stain and an ancient tooth sample. However, working with ancient DNA requires additional cloning and sequencing of the RFLP-typing results due to DNA post mortem damages such as deaminations, which could lead to false typing results.     

The incidence and features of serological ABO subgroups among one million blood donors in Beijing

This study aims to determine the incidence of serological ABO subgroups from a large-scale database, along with the features of blood samples with serological ABO discrepancies. The serological ABO results of one million individuals were randomly sampled from a blood donor database in Beijing between 2009 and 2010. All samples were diagnosed by serological reverse and forward ABO typing using an automatic analyzer. The proportions of the normal ABO types were 27.28%, 31.57%, 30.56%, and 10.16% for blood types A, B, O, and AB, respectively. In samples in which ABO discrepancies or obvious weak agglutinin were identified in the forward or reverse typing, further tests to analyze the ABO subgroup were conducted. The overall incidence of ABO subgroups was 0.047%, with 14 ABO subgroups observed: A2, A3, Ax, Am, Aint, Aend, B2, B3, Bx, Bm, Bel, B(A), cisAB, and AB^h. In conclusion, this study revealed the exact normal ABO and subgroup distributions in the general, healthy population of Beijing using samples from a blood donor database.     

Consistency of ABO blood group phenotypes and genotypes in renal transplant patients

The aim of this study was to confirm the concordance between the ABO phenotype and genotype in 34 patients undergoing renal transplant before 2010 in Sir Run Run Shaw Hospital. The ABO genotyping kit and column agglutination test (CAT) were used to examine the ABO type, and ABO subgroup was checked by sequence analysis of ABO exons 6 and 7. We found that the genotypes of serological A, AB, O, and B patients were A1A1 in 3 patients and A1O1 in 5 patients, A1B, O1O2 in 1 patient and O1O1 in 11 patients, and BB in 6 patients and BO1 in 6 patients, respectively. However, one patient, who was originally reported as serological B in the 2010 medical record and CAT showed Asub B in 2016 and sequence analysis of ABO exons 6 and 7 demonstrated B(A)04/O1.[not clear] The ABO column agglutination testing combined with genotyping may provide additional value in pre-renal transplantation laboratory examinations, and it may be safe to transplant a B/O1 kidney to a B(A)04/O1 recipient since the transplantation has been success for 6 years.     

The functional A allele was resurrected via recombination in the human ABO blood group gene

Functional A and B alleles are distinguished at two critical sites in exon 7 of the human ABO blood group gene. The most frequent nonfunctional O alleles have one-base deletion in exon 6 producing a frameshift, and it has the A type signature in two critical sites in exon 7. Previous studies indicated that B and O alleles were derived from A allele in human lineage. In this study, we conducted a phylogenetic network analysis using six representative haplotypes: A101, A201, B101, O01, O02, and O09. The result indicated that the A allele, possibly once extinct in the human lineage a long time ago, was resurrected by a recombination between B and O alleles less than 300,000 years ago.     

运用多重PCR-直接测序法检测ABO基因型及其遗传多态性

根据ABO基因座第6和7外显子9个SNP位点设计引物, 复合扩增后直接测序, 根据测序结果判定不同物证检材ABO基因型及其在藏族群体中的多态性分布。成功地检测出经过不同方法处理的血痕、毛发、口腔拭子、骨骼、混合斑等101例腐败、降解及微量检材的ABO基因型, 结果与免疫血清学分型一致, 且该方法具有灵敏度高、特异性好、操作简单、结果准确、客观及能够发现新等位基因等优点。对80名青海藏族无关个体的调查表明, ABO基因型分布符合Hardy-Weinberg平衡, 杂合度H为0.675, 多态信息含量PIC为0.672, 个人识别力DP值为0.874, 非父排除率PE值为0.391, 偶合度I为0.126; 青海藏族ABO等位基因频率O>B>A, 且O等位基因频率高达0.6125。多重PCR-直接测序法检测ABO基因型适用于法医学不同来源的样本, 提高了ABO血型系统的个体识别能力; ABO基因型在青海藏族人群中的分布具有较高多态性, 可用于法医学个体识别及群体遗传学研究。     

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.     

Molecular analysis to FUT-1, 2, 3 and ABO genotyping may instead of serological typing to diagnose para-Bombay in Chinese

The aim of this study was to evaluate the consistency between serotyping and molecular analysis in Chinese with para-Bombay. The molecular analysis of gene fragments in FUT-1, FUT-2, FUT-3 and ABO genotyping and serotyping were used including a saliva test to examine the A, B, H substance and an absorption elution test to examine the A, B, H; and further routine tests including ABO, H and Lewis phenotype. From eleven samples with anti-H negative, 10 samples were confirmed with para-Bombay by sequencing to FUT-1, from which six samples were 547-548delAG, three samples were 880TT deletion, one sample was 35C>T and one sample was 649G>T heterozygous (h7, China) as carrier. The sequencing to FUT-2 confirmed 357C>T in 11 samples, meaning H, A and B substance was secreted in saliva except for one sample which occurred 385A>T (I129F) heterozygous, which is a weak secretor. The FUT-3 sequence result demonstrated four samples with heterozygous mutations to 59T>G (L20R) combined with 508G>A (G170S) and seven samples without mutations in FUT-3 gene fragment same as reference. The consistency between sequencing with FUT-1/FUT-2 and serotyping by anti-H reported an identical result, except for one sample, which interestingly showed the H/h7 carrier with serotyping negative to anti-H. The result of sequencing with FUT-2/FUT-3 and Lewis phenotyping also reported a complete consistency. The saliva test to A, B, H substance and absorption elution test examining the A, B, H antigens on the surface of red blood cells completely matched the ABO exon 6, 7 sequence results. The sequencing of FUT-1, FUT-2, FUT-3 and ABO exon 6, 7 may become a useful tool to confirm the para-Bombay blood type.     

A de novo recombination in the ABO blood group gene and evidence for the occurrence of recombination products

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio’s genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene. Received: 25 October 1996     

Absorption of anti-blood group A antibodies on P-selectin glycoprotein ligand-1/immunoglobulin chimeras carrying blood group A determinants: core saccharide chain specificity of the Se and H gene encoded alpha1,2 fucosyltransferases in different host cells

To specifically eliminate recipient anti-blood group ABO antibodies prior to ABO-incompatible organ or bone marrow transplantation, an efficient absorber of ABO antibodies has been developed in which blood group determinants may be carried at high density and by different core saccharide chains on a mucin-type protein backbone. The absorber was made by transfecting different host cells with cDNAs encoding a P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) chimera (PSGL-1/mIgG(2b)), the H- or Se-gene encoded alpha1,2-fucosyltransferases (FUT1 or FUT2) and the blood group A gene encoded alpha1,3 N-acetylgalactosaminyltransferase (alpha1,3 GalNAcT). Western blot analysis of affinity-purified recombinant PSGL-1/mIgG(2b) revealed that different precursor chains were produced in 293T, COS-7m6, and Chinese hamster ovary (CHO)-K1 host cells coexpressing FUT1 or FUT2. FUT1 directed expression of H type 2 structures mainly, whereas FUT2 preferentially made H type 3 structures. None of the host cells expressing either FUT1 or FUT2 supported expression of H type 1 structures. Furthermore, the highest A epitope density was on PSGL-1/mIgG2(2b) made in CHO-K1 cells coexpressing FUT2 and the alpha1,3 GalNAcT. This PSGL-1/mIgG(2b) was used for absorption of anti-blood group A antibodies in human blood group O serum. At least 80 times less A trisaccharides on PSGL-1/mIgG(2b) in comparison to A trisaccharides covalently linked to macroporous glass beads were needed for the same level of antibody absorption. In conclusion, PSGL-1/mIgG(2b), if substituted with A epitopes, was shown to be an efficient absorber of anti-blood group A antibodies and a suitable model protein for studies on protein glycosylation.     

Molecular genetic analysis of weak B blood group allele in Jinan population reveals distribution of ABw phenotype

The aim of this study was to investigate the distribution of the ABw phenotype of ABO blood group in the Jinan population. 31 856 samples were tested during the period 2018 to 2019. Thirty-nine samples with discrepant results, as identified by micro-column gel method, were further investigated by serological (tube technique) and molecular (fluorescence PCR, DNA sequencing) methods. Eight samples showed ABw phenotype, which accounted for 0.025% of the population tested. From the sequencing analysis, six samples (6/8) were typed as ABO*A1.02/ABO*BW.12 and two samples (2/8) as ABO*A1.02/ABO*BW.03. The study suggests that ABw12 account for 75% of ABw phenotype and indicate ABw12 is the main ABw phenotype in Jinan population.     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Serological and molecular analysis of ABO and Rh blood group chimeras

The serological examination, blood transfusion strategies and the molecular analysis to blood group chimera were conducted to demonstrate existent of chimera in blood group. The blood grouping of ABO or/and RhD, newborn red blood cells separated by capillary centrifugation. Aabsorption tests and DTT treated agglutination erythrocyte tests were implemented in four patients. Further molecular biological research was conducted on one patient''s sample. The results showed that for patient 1: ABO blood group was AB/B chimera, Rh blood cells contained the RhCE chimera gene; Patient 2: Rh blood cells contained the RhD chimera gene; Patient 3: ABO blood group was AB/B chimera, Rh blood cells contained the RhD chimera gene; Patient 4: ABO blood group was O/B chimera, Rh blood cells contained the RhCE chimera gene. The study suggests that the individuals categorized as chimeras are likely to be more common than existing literature reports. According to the serological tests, in the absence of a history of recent blood transfusion or disease to cause reduced antigen, the phenomena of hybrid aggregation of the ABO and Rh blood system were the main feature. In terms of transfusion strategy, the selection of ABO and Rh blood groups should be depended on the group of cells with more antigens.