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 a novel mutation of B glycosyltransferase gene related with an ABx variant].

OBJECTIVE: To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system. METHODS: Serological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene. RESULTS: Erythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B. CONCLUSION: The 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

[Study on polymorphism of membrane glycoprotein genes related to human platelet alloantigens].

OBJECTIVE: To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w. METHODS: The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author's designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms. RESULTS: Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/A and 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPIIIa. CONCLUSION: New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.

[Molecular mechanism of an individual with weaken B phenotype in ABO blood group].

OBJECTIVE: To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen. METHODS: The ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5'-untranslated region (5'-UTR) sequence of ABO gene were amplified by polymerase chain reaction (PCR) respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR(RT-PCR) and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method. RESULTS: The serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B glycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5'-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype, a number of methylated CpG sites were found near the promoter of ABO gene in this sample. CONCLUSION: The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.

[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.

[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.

[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.

[Polymorphism of killer cell immunoglobulin-like receptor 2DL3 gene in Zhejiang Han population].

OBJECTIVE: To analyze the allelic polymorphism of killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3) gene in Zhejiang Han population. METHODS: Allelic of KIR2DL3 was detected by PCR sequence based typing (PCR-SBT) method. Two specific fragments of KIR2DL3 were amplified using different primers and sequenced for exons 4,5,7,8,9 of KIR2DL3. All the samples would be assigned to KIR2DL3 allele according to the polymorphism site patterns. RESULTS: Two KIR2DL3 alleles were observed, with KIR2DL3*001 having higher frequency, 0.77. CONCLUSION: The PCR-SBT method is reliable and there are distinctive frequencies of KIR2DL3 alleles in Zhejiang Han population.

[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.

[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.

[Polymorphisms of six short tandem repeat loci in Zhejiang She ethnic population and Zhejiang Hans of China].

OBJECTIVE: To investigate genetic polymorphism of six short tandem repeat (STR) loci (D3S1358,D16S539, TH01,TPOX, CSF1PO,D7S820) in Zhejiang She ethnic population and Han population. METHODS: By use of AmpFlSTR Cofiler kit, 6 STR loci in 108 She samples and 102 Han samples were amplified. The PCR products were electrophoresed by ABI Prism 377 sequencer; the data were analyzed by Genescan software. RESULTS: All genotype frequencies of the 6 STR loci in She and Han ethnic groups met Hardy-Weinberg equilibrium. In the She population, the heterozygosities (H) in D3S1358,D16S539,TH01,TPOX, CSF1PO and D7S820 were 0.8028, 0.9148, 0.7522, 0.6728, 0.9123,0.8338, the exclusion of paternity(EP) were 0.4067, 0.6057, 0.4437, 0.3200, 0.5250, 0.5358, discrimination power (DP) were 0.6690, 0.7841, 0.6447, 0.5382, 0.7298, 0.7296.The combined DP, PE and polymorphism information content were 0.9991,0.9805,0.9988 respectively. There were significant differences at D3S1358, D16S539 and TPOX loci, compared with Hans. CONCLUSION: She population has its own STR allele distribution characteristic. The above data obtained from She population can be used not only in genetic researches and population investigation, but also in human identity and paternity testing.

[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.

[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.