To investigate the frequency of RHD 1227A allele in Rh negative population and random population, an AS-PCR (allele specific-polymerase chain reaction) method was employed to detect RHD 1227A allele. RHD gene copy was determined by D zygosity test and RHD exon 9 nucleotide sequence analysis. The results showed that among 143 Rh negative donors, forty-one RHD 1227A allele carriers were detected, and 8 (19.51%) out of which were RhCCdee, 32 (78.05%) were RhCcdee, and 1 (2.44%) was RhCcdEe. Thirty-five Rh negative RHD 1227A carriers had RHD gene deletion, and the remaining carriers were RHD 1227A homozygous. Seven (1.43%) individuals were detected with RHD 1227A allele among 489 random donors. They were all G/A heterozygous at RHD 1227 site. Serological test indicated that they were normal Rh positive phenotype. It is concluded that the frequency of RHD 1227A allele is 16.43% among Rh negative population and 0.72% among the random population.
Background Although many molecular diagnostic methods have been used for ABO genotyping, there are few reports on the full-length genomic sequence analysis of the ABO gene. Recently, next-generation sequencing (NGS) has been shown to provide fast and high-throughput results and is widely used in the clinical laboratory. Here, we established an NGS method for analyzing the sequence of the start codon to the stop codon in the ABO gene. Study Design and Methods Two pairs of primers covering the partial 5’-untranslated region (UTR) to 3’-UTR of the ABO gene were designed. The sequences covering from the start codon to the stop codon of the ABO gene were amplified using these primers, and an NGS method based on the overlap amplicon was developed. A total of 110 individuals, including 88 blood donors with normal phenotypes and 22 ABO subtypes, were recruited and analyzed. All these specimens were first detected by serological tests and then determined by polymerase chain reaction sequence-based typing (PCR-SBT) and NGS. The sequences, including all the intron regions for the specimens, were analyzed by bioinformatics software. Results Among the 88 blood donors with a normal phenotype, 48 homozygous individuals, 39 heterozygous individuals, and one individual with a novel O allele were found according to the results of the PCR-SBT method. Some single-nucleotide variants (SNV) in intronic regions were found to be specific for different ABO alleles from 48 homozygous individuals using the NGS method. Sequences in the coding region of all specimens using the NGS method were the same as those of the PCR-SBT method. Three intronic SNVs were found to be associated with the ABO subtypes, including one novel intronic SNV (c.28+5956T>A). Moreover, six specimens were found to exhibit DNA recombination. Conclusion An NGS method was established to analyze the sequence from the start codon to the stop codon of the ABO gene. Two novel ABO alleles were identified, and DNA recombination was found to exist in the ABO alleles.
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.
Objective To investigate the serological characteristics and molecular basis of the B (A)phenotype in ABO blood group and provide the data for clinical transfusion of individuals with B(A) phenotype.Methods The ABO group antigens on red cells of the proband,family members and donors were identified by monoclonal antibodies and the ABO antibodies in sera were detected by the standard A,B,O cells.The compatibility testing for the proband and donors was detected by salted test,polybrene test and antiglobulin test.The coding region of exon 6 to exon 7 in ABO gene was amplified by polymerase chain reaction(PCR) and the PCR products were sequenced.The haplotypes of proband were analyzed by cloning and sequencing.Results It was showed that both A and B antigens were detected on red cells of the proband and her two family members,and there was anti-A_1 antibody in their sera.The serological phenotype of the samples are identified as the A_2B.DNA sequencing showed 261 G/del,297A/G,526C/G,657C/T,700C/G,703G/A,796C/A,803G/C,930G/A heterozygotes in exon 6 to exon 7.It can be deduced that genotype in the proband is B(A)_(02)/O_(01).The genotypes of her mother and grandmother-in-law were B(A)_(02)/B_(101) and B(A)_(02)/O_(01),respectively.After cloning and sequencing,two alleles B(A)_(02) and O_(01) in proband was showed.B(A)_(02) has snigle nucleotide change(700 C>G),which resets replacement of proline with alanine at position 234.Two donors with phenotype A_2B were identified as genotype B(A)_(02)/O_(01) and A_(208)/B_(101),respectively.The results of crossmatch testing is in accordane between the proband and two donors and there was no clinical adverse reaction after transfusion.Conclusions 700C>G in α-1,3galactosyltransferase allele(B allde)can result in B(A)phenotype in individuals with the phenotype of A_2B.The donors in the transfusion for the individuals with B(A) phenotype should include individuals with A_2B phenotype.
Key words:
ABO blood-group system; Blood group incompatibility; Pedigree
To investigate the molecular genetic basis of the B3 variant of ABO blood group system with mixed-field hemagglutination in Chinese.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.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.A novel B allele with 425T>C substitution resulting in B3 subgroup was identified in two Chinese individuals.
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