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To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.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.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 808TA mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B.The 808TA mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype.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.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.G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.

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.

To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband.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.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.A novel B112 allele of ABO group system with 559CT was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

To investigate the molecular genetic basis of para-Bombay phenotype in a Chinese family.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.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.A novel 235GC mutation of FUT1 gene which was associated with para-Bombay phenotype was found in the Chinese pedigree.

To analyse the reason for one case of hemolytic transfusion reaction, antibodies in a patient's serum were identified using panel cells and Le (a-b-) phenotype cells, patient phenotype was identified by using anti-Le(a) and anti-Le(b) blood grouping reagents and the entire coding region of FUT3 gene was amplified by PCR and sequenced directly. The results showed that both IgM anti-Le(a) and anti-Le(b) antibodies were detected in patient's serum. Red cells was typed as Le (a-b-) phenotype and the FUT3 genotype was homozygote for non-functional le(59, 508) alleles. In conclusion, anti-Le(b) antibody can result in hemolytic transfusion reaction, FUT3 gene is homozygous for le(59, 508) allele resulting in Le (a-b-) phenotype.

In order to establish a genotyping method for DEL phenotype in Zhejiang Han population, an AS-PCR method was developed according to the RHD 1227A allele sequence. Its specificity and sensitivity were assessed in two Rh negative populations whose RHD 1227A or DEL phenotype status was known. The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method. After re-testing these two samples with serological method and sequence analysis, it was found that original serological method gave false negative results and genotyping method still showed 100% specificity. The minimal target DNA concentration of this genotyping method is 8.13 ng/microl. In conclusion, designed genotyping method can be used to identify DEL phenotype efficiently in Zhejiang Han Rh negative population.

The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467CT and 539GC) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.

To investigate the alpha-1, 3/4-fucosyltransferase gene (FUT3) polymorphism associated with Lewis blood group in Zhejiang population, the Lewis phenotypes of 183 random samples from Chinese blood donors in Zhejiang province were identified by standard serological techniques. The entire coding region of FUT3 gene were amplified by PCR from genomic DNA of 39 Lewis negative and 9 Lewis positive phenotype samples and sequenced directly. The haplotypes of FUT3 allele were identified by TOPO cloning sequencing method. The results showed that the frequency of true Le (a-b-) phenotype in Zhejiang population was 10.4% according to serological and molecular biological methods. Five nucleotide acid variant sites (59TG, 202TC, 314CT, 508GA and 1067TA) were detected in all 48 sequencing samples. Besides the wild type Le allele, 2 common (le(59, 1067) and le(59, 508) and 3 rare non-functional le alleles (le(59), le(1067) and le(202, 314) were found in this population. In conclusion, the polymorphism of non-functional FUT3 allele was found to be relatively variable in Chinese Zhejiang population.

To investigate the molecular genetics basis for one para-Bombay phenotype, the red blood cell phenotype of the proband was characterized by standard serological techniques. Exon 6 and 7 of ABO gene, the entire coding region of FUT1 gene and FUT2 gene were amplified by polymerase chain reaction from genomic DNA of the proband respectively. The PCR products were purified by agarose gels and directly sequenced. The PCR-SSP and genescan were performed to confirm the mutations detected by sequencing. The results showed that the proband ABO genotype was A(102)A(102). Two heterozygous mutations of FUT1 gene, an A to G transition at position 682 and AG deletion at position 547-552 were detected in the proband. A682G could cause transition of Met--Val at amino acid position 228, AG deletion at position 547-552 caused a reading frame shift and a premature stop codon. The FUT2 genotype was heterozygous for a functional allele Se(357) and a weakly functional allele Se(357), 385 (T/T homozygous at position 357 and A/T heterozygous at 385 position). It is concluded that the compound heterozygous mutation--a novel A682G missense mutation and a 547-552 del AG is the molecular mechanism of this para-Bombay phenotype.

To investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population.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.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.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.