Molecular Basis of ABO Variants Including Identification of 16 Novel ABO Subgroup Alleles in Chinese Han Population.

INTRODUCTION: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion. METHODS: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. RESULTS: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. CONCLUSIONS: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

[Study on the expression stability of mutant alpha-1,2 fucosyltransferase gene 293C to T and 658C to T in eukaryotic cells].

OBJECTIVE: To establish the eukaryotic cell expression system for the alpha-1,2 fucosyltransferase gene FUT1 293C to T and 658C to T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen. METHODS: Genomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified. The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography. Expressed protein was identified by SDS-PAGE and Western blotting. RESULTS: pcDNA3.1-FUT1 wildtype, pcDNA3.1-FUT1 293C to T and pcDNA3.1-FUT1 658C to T recombinant vectors were constructed, respectively. COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418. The FUT1 mRNA level of transfected cells with 293C to T and 658C to T recombinant vectors reached 97.10% and 104.74% of the wildtype FUT1 transfected cell. A specific protein band with about 46 000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6×His Tag antibody. The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction, while the enzyme activity of cell lysates from 293C to T and 658C to T were abolished. CONCLUSION: The results suggested that the 293C to T and 658C to T mutations of FUT1 gene did not affect the RNA and protein expression levels, but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.

[Identification of an ABx09 phenotype of ABO subtype].

OBJECTIVE: To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype. METHODS: The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies, and the ABO antibody in serum was detected by standard A, B, O cells. The exons 1 to 7 of ABO gene were amplified by polymerase chain reaction (PCR) respectively and the PCR products were sequenced directly. The amplified products for exons 5 to 7 were also cloned by TOPO TA cloning sequencing kit to split the two alleles apart, selected colonies were sequenced bidirectionally for exons 5 to 7 of the ABO gene. The samples of the proband's parents were collected, then serological test of the blood group and sequence analysis for exons 6 and 7 of ABO gene were preformed. RESULTS: Both A and B antigens were detected on red blood cells of the proband and there was anti-B antibody in the serum. There was no G deletion at position 261, while 297AG in exon 6, 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 889GA and 930GA heterozygote in exon 7 were detected by direct DNA sequencing, which can be assigned for A102Bx09 genotype. After cloning and sequencing, two alleles A102 and Bx09 were obtained. The sequence of Bx09 had one nucleotide changes (G to A) at position 889 compared with that of B101, which resulted in an amino acid change of Glu to Lys at 297 position. The Bx09 in the proband was inherited from her mother by family investigation. CONCLUSION: G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.

[Cloning and sequence analysis of 5'untranslated region of human ABO gene].

OBJECTIVE: To clone and investigate the polymorphism of the 5'-untranslated region (5'-UTR) of the human ABO gene, in order to provide the basis for exploring the transcriptional regulation of the human ABO histo-blood group genes. METHODS: ABO phenotypes of 30 unrelated healthy blood donors were determined by serological technique, their genotypes were analyzed by sequencing the exons 6 and 7 of the ABO gene. Nearly 5 kb of the 5'-UTR of ABO gene was obtained by PCR amplification and sequencing was performed bidirectionally. Haplotypes of samples with heterozygous sites in the 5'-UTR of ABO gene were separated and analyzed after cloning. RESULTS: Twenty polymorphic sites were identified in these samples where ABO genotypes were consistent with serological phenotypes. It included sixteen nucleotide sequence variations, one 8 bp deletion, one 6 bp deletion/insertion, one 36 bp insertion and one 43 bp repeats. Among them, 11 were novel polymorphic sites. Seven different haplotypes of 5'-UTR of ABO gene were defined to the cis/trans linkage of those mutations. CONCLUSION: There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.

[Molecular genetic analysis of a new B112 allele of ABO blood group].

OBJECTIVE: To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband. METHODS: The ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed. RESULTS: The serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband. CONCLUSION: A novel B112 allele of ABO group system with 559C>T was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

[Analysis of alpha-1,2-fucosyltransferase gene mutations in a Chinese family with para-Bombay phenotype].

OBJECTIVE: To investigate the molecular genetic basis of para-Bombay phenotype in a Chinese family. METHODS: ABO and H phenotypes of the proband and his pedigree were characterized by serological techniques. The exons 6 and 7 of the ABO gene and full coding region of alpha-1,2-fucosyltransferase (FUT1) gene of the pedigree were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotypes of compound heterozygote of the FUT1 gene were also analyzed by cloning sequencing. RESULTS: Three para-Bombay phenotypes were identified in nine family members by serological technology. Three heterozygous variants (35C/T, 235G/C and 682A/G) were found in FUT1 gene of the proband, and the hapotype of FUT1 gene was h(235C)/h(35T+628G)according to the cloning sequencing. The alleles h(235C)and h(35T+628G) caused G79R, A12V and M228V amino acid substitutions in alpha-1,2-fucosyltransferase, respectively. CONCLUSION: A novel 235G>C mutation of FUT1 gene which was associated with para-Bombay phenotype was found in the Chinese pedigree.

[A novel M142T mutation in the B glycosyltransferase gene associated with B3 variant in Chinese].

OBJECTIVE: To investigate the molecular genetic basis of the B3 variant of ABO blood group system with mixed-field hemagglutination in Chinese. METHODS: Serological techniques were performed to characterize the erythrocyte phenotype of two discrepant samples. A sequential agglutination method and 13 short tandem repeat (STR) loci were tested to exclude the possibility of exogenous or endogenous DNA chimera. Mutations in exons 6 and 7, including partial intron of the ABO gene, were screened by polymerase chain reaction and DNA sequencing. Haplotypes of the two individuals were also analyzed by sequencing. RESULTS: A mixed-field hemagglutination of RBCs with anti-B and anti-AB antibodies was detected in the two unrelated individuals. Exogenous ABO-incompatible RBC transfusion and endogenous genetic chimera were excluded by sequential agglutination method and STR. The ABO phenotypes of the two individuals were classified as A1B3 according to the ABO subgroup definition. The sequence region from intron 5 to 3'-UTR of the B allele was identical to that of ABO*B101 allele, except for a T to C substitution at nucleotide position 425 in exon 7. This substitution resulted in an amino acid change of M142T in the B glycosyltransferase. CONCLUSION: A novel B allele with 425T>C substitution resulting in B3 subgroup was identified in two Chinese individuals.

[Molecular genetic analysis of rare cisAB variants in Chinese population.].

We investigated the molecular genetic basis of rare cisAB variants at the ABO locus in Chinese population. Serological techniques were performed to characterize erythrocyte phenotype of 12 discrepant samples and 116 randomly selected samples. Mutations of complete exon 6 and 7 including flanking intron in the ABO genes were screened by PCR and directly sequencing. The haplotypes of diverse genotypes were also analyzed by cloning sequencing. Twelve samples were identified as AweakB or ABweak phenotype by serological technology, and were also identified as cisAB variants including four genotypes by directly sequencing. Two cisAB alleles were found by haplotype sequencing. One allele was cisAB01, in which four nucleotide acid alterations were observed (467C>T and 803G>C in exon 7, 163T>C and 179C>T in intron 6); the other allele maintained a nucleotide acid of A101 allele (803G) compared with B101 allele, which resulted in a polypep-tide containing 176G, 235S, 266M, and 268G four amino acids. This is a novel allele, which has been named cisAB05 by Blood Group Antigen Gene Mutation Database. According to systematically investigation of the molecular genetic basis of the cisAB variants in Chinese population, we found a novel cisAB05 allele and presumed that the cisAB01 allele is derived from homologues exchange of A101-B101 combination.

[278C > T variant of the alpha-1, 3-galactosyltransferase allele responsible for Bw subgroup].

OBJECTIVE: To investigate the molecular genetic basis of the Bw variant and identify novel alleles at ABO locus in Chinese Han population. METHODS: Serological techniques were performed to characterize erythrocyte phenotype of a proband. Mutations of the ABO gene were screened by polymerase chain reaction, reverse transcription-polymerase chain reaction and DNA sequencing. RESULTS: The proband was identified as Bw phenotype by serological technology and family study. A novel Bw variant allele was identified in the gDNA and cDNA. The novel allele was observed a missense mutation (278 C to T) at the exon 6 which resulted in an amino acid substitution (P93L) compared with B101 allele. The 278 C to T was the first report mutation position in exon 6 among Bw alleles, so the P93L amino acid substitution was different from others Bw variants which had amino acid substitutions in a conserved functional domain reported previously. CONCLUSION: A novel Bw allele (278 C to T) responsible for Bw variant is reported in Chinese population.

[Identification of nine novel alternative splicing isoforms of RHD mRNA].

To investigate the gene structures of RHD mRNA alternative splicing isoforms, total RNA was extracted from normal cord blood samples and RHD mRNA were detected using reverse transcription and polymerase chain reaction (RT-PCR) method. The PCR products were cloned and directly sequenced. The splicing signals of various isoforms were also analyzed by sequencing of exon-intron boundaries using RHD gene specific primers, and the expressed sequence tags (ESTs) databank was screened to find out other RHD alternative splicing isoforms. Nine novel and three recurrent RHD alternative splicing isoforms were identified, among which six isoforms were RHD-RHCE hybrid genes. Three types of alternative splicing were found including exon skipping, alternative 5' splice sites and alternative 3' splice sites. Exons 2 to 9 were involved in RHD mRNA alternative splicing. Two other isoforms of intron retention type were also found in human ESTs databank. Thus, the mRNA splicing mechanism of RHD gene was very complex, and nine novel alternative splicing isoforms were identified including several isoforms based on homologous RHD-RHCE hybrid gene.

[Study on the frequency of alpha-1,2-fucosyltransferase gene h4 allele (C35T) in Chinese population].

OBJECTIVE: To investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population. METHODS: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying C35T variant was established by using PCR to amplify a 125 bp FUT1 gene fragment, including C35T variant sequence, and the PCR product was digested by Hae III restriction enzyme. One hundred and fifty-eight random samples from Chinese blood donor were screened by PCR-RFLP. RESULTS: Among 158 Chinese individuals with normal ABO and H blood group phenotypes, 8 and 83 were homozygous with 35T/T and 35C/C, respectively, while 67 were heterozygous with 35C/T. The allele frequencies were compatible with Hardy-Weinberg equilibrium. CONCLUSION: The C35T substitution of FUT1 gene is not a mutation which gives rise to a non-functional h allele responsible for para-Bombay phenotype but a single nucleotide polymorphism in Chinese population.

[Analysis of Human Platelet Antigen-1 System Alloantibodies Using Recombinant GPIIIa Fragments Coupled to Luminex Beads].

OBJECTIVE: To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads. METHODS: The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop. RESULTS: The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies. CONCLUSION: The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

[One base deletion of the alpha(1,4) galactosyltransferase gene responsible for p phenotype].

OBJECTIVE: To study the molecular genetic basis for p phenotype. METHODS: Red blood cell phenotype of the proband was characterized by standard serological technique. Exon 3 of the alpha (1,4) galactosyltransferase gene was amplified by polymerase chain reaction from genomic DNA of the proband. The amplified PCR products were excised and purified from agarose gels and direct sequenced. RESULTS: Homozygous single nucleotide G deletion at position 300 or 301 was found in the proband, which caused a reading frame shift at codon 101,resulting in a premature stop at codon 113. Parents of the proband were heterozygous carriers. CONCLUSION: A novel single guanosine deletion at position 300 or 301 of alpha (1,4) galactosyltransferase gene was determined, which may be one facet of the molecular basis for p phenotype.

[Two base deletion of the alpha (1,2) fucosyltransferase gene responsible for para-Bombay phenotype].

OBJECTIVE: To probe into the molecular genetics basis for para-Bombay phenotype. METHODS: Red blood cell phenotype of the proband was characterized by serological techniques. Exons 6 and 7 of ABO gene, the entire coding region of alpha(1,2) fucosyltransferase (FUT1) gene and FUT2 gene were amplified by polymerase chain reaction (PCR) from genomic DNA of the proband respectively. The PCR products were excised and purified from agarose gels and were directly sequenced. RESULTS: AG at 547-552 deletion homozygous allele was found in the proband, which caused a reading frame shift and a premature stop codon. Parents of proband were heterozygous carriers. CONCLUSION: Two base deletion at position 547-552 of alpha (1,2) fucosyltransferase gene may cause para-Bombay phenotype.

[Establishment of method detecting CD36 expression on human platelet and its application].

The individual with the deficiency of CD36 antigen on platelet displayed the risk of anti-CD36 immune reaction induced by transfusion, which is one of the reasons for platelet transfusion refractoriness (PTR). This study was purposed to detect the expression level of CD36 antigen on platelet by flow cytometry among apheresis platelet donors of Hangzhou area, and the frequency of CD36 deficiency was analyzed. Platelet-rich plasma (PRP) was separated from fresh anticoagulant whole blood by centrifugation, then the platelets were washed and adjusted to 1×10(6). The platelets were incubated with FITC-labeled CD36 and PE-labeled CD41 monoclonal antibodies, then the expression level of CD36 was detected by flow cytometry. The CD36 expression on monocytes for the samples of CD36-deficiency on the platelets was further analyzed. The results showed that 7 samples with CD36 antigen deficiency were found in 192 apheresis platelet donors. The frequency of CD36 deficiency was 3.6% and all of them were typeII deficiency. The significant difference of CD36 antigen expression was observed in the platelet donors of Hangzhou population, among them 59 individuals with low expressed CD36 antigen and 126 individuals with highly expressed CD36 antigen were found according to the geometric mean fluorescence intensity. It is concluded that the CD36 antigen deficient phenotype existed in the population, these data will provide the information for research of the CD36 antigen distribution and help to solve the platelet transfusion refractoriness.

[Detection of hematopoietic chimera by real-time fluorescent quantitative PCR with erythrocyte Kidd blood group gene].

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.

[Rare blood group screening by serological and molecular methods in Zhejiang Han population].

This study was aimed to investigate the distribution of rare blood group in Zhejiang Han population. The H(-) (H system), GPA(-) and s(-) (MNS), Rhnull, Rhmod, D--, CCDEE, CCdEE (variations of Rh), GPC(-) (Gerbich), i(+) (I), Lu(b-) (Lutheran), Js(b-) and k(-) (Kell), Fy(a-) (Duffy), Ok(a-) (Ok), Di(b-) (Diego) phenotypes were screened by serological or molecular methods. Jk (a-b-) phenotype was detected by urea hemolytic test. The results showed that one Di (a+b-) individual was found in 1618 blood donors, three Fy (a-b+) individuals in 1007 donors and one CCdEE individual in 633 Rh negative donors. No Jk (a-b-), H(-), GPA(-), s(-), GPC(-), i(+) (adult), Lu(b-), k(-), Js(b-), Lu(b-) and Ok(a-) phenotypes were found in this large scale survey. It is concluded that Di (a+b-), Fy (a-b+), CCdEE phenotypes are confirmed in the blood donors and this study provides the distribution data of erythrocyte rare blood group in Zhejiang Han population.

[Para-Bombay phenotype caused by combined heterozygote of two bases deletion on fut1 alleles].

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.

[C742T mutation of α1, 3 N-acetyl-D-galactosaminyltransferase gene is responsible for A2 subgroup].

The objective of this study was to analyze the molecular genetic basis for 2 individuals with A2B phenotype of ABO subtype. The ABO group antigens on red blood cells were identified by monoclonal antibodies and the ABO antibodies in serum were detected by the standard A, B, O cells. The exon 5 to exon 7 coding region of ABO gene was amplified by polymerase chain reaction (PCR) and the PCR product was sequenced directly after the enzymes digested. The amplified product was also cloned by TOPO TA cloning sequencing kit to split the 2 alleles apart and chosen colonies were sequencing bidirectionally for exon 6 to 7 of ABO gene. The results showed that both A and B antigen were identified on red blood cells of the individuals and there was anti-A1 antibody in their serum. There was no 261G deletion and showed 297A/G, 467C/T, 526C/G, 657C/T, 703G/A, 742C/T, 796C/A, 803G/C, 930G/A heterozygotes by direct DNA sequencing. After cloning and sequencing, it was obtained the B101 and one novel A allele. The novel allele has one nucleotide change at 742 position C to T compared with A102, which results in an amino acid Arg to Cys at 248 position and was nominated as A213. It is concluded that C742T mutation of the α1, 3 N-acetyl-D-galactosaminyl-transferase gene can lead to A2 phenotype and with anti-A1 antibody in serum.