[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.

[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.

[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.

[Probability of high resolution full match for human leukocyte antigen loci in unrelated donors and recipients with low resolution match].

This study was aimed to analyze the possibility of high resolution matching for human leukocyte antigen (HLA) loci in unrelated donor-recipient pair with low resolution match in HLA-A, -B, -DRB1 loci. Samples were genotyped for HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reaction sequence based typing (PCR-SBT). The results showed that the total number of patients and the donors were 166 and 274. 97 (58.43%) patients were matched for 1 donor and 47 (28.31%) patients were matched for 2 donors at low resolution level; among 274 donor-recipient pairs, HLA-A, -B, -C, -DRB1 and -DQB1 loci matching for 6/10, 7/10, 8/10, 9/10 and 10/10 were 32 (11.68%), 54 (19.71%), 62 (22.63%), 49 (17.88%) and 48 (17.52%) respectively; there were mismatch in HLA-A, -B, -C, -DRB1 and -DQB1 loci, and the most mismatch was in HLA-C locus. The number of alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were 23, 46, 21, 30 and 17 respectively in the donors. The alleles number HLA-A, -B, -C, -DRB1 and -DQB1 loci were 20, 40, 22, 29 and 16 respectively in the patients; the haplotype number of HLA loci were 311 in the donors and 224 in the patients. The high frequency of haplotype was A*02:07-B*46:01-C*01:02-DRB1*09:01:02-DQB1*03:03 (5.63% and 6.88%). It is concluded that the probability of high resolution mismatch of HLA loci is high in unrelated donor-recipient pairs with low resolution match in HLA-A, -B, -DRB1 loci.

[Establishment of allele specific primer polymerase chain reaction sequence-based typing and investigation of the polymorphism in exon 3 of HLA-DRB1].

OBJECTIVE: To establish the allele specific primer polymerase chain reaction sequence-based typing (PCR-SBT) and investigate the polymorphism of exon 3 of human leukocyte antigen( HLA)-DRB1. METHODS: The fragment containing exons 2 and 3 of HLA-DRB1 gene was amplified by group specific primers. The amplified products were digested by restriction enzymes and directly sequenced in both directions. The genotype was assigned by using Assign 3.5 SBT software. RESULTS: The exon 3 sequences of HLA-DRB1*08:09 and HLA-DRB1*12:02:01 were identified for the first time. There were 27 polymorphic sites in exon 3 among the twenty-five HLA-DRB1 alleles, which was 9.56% of all nucleotides of exon 3. The method could discriminate the HLA-DRB1*14:01:01/14:54 ambiguous samples, and the HLA-DRB1*14:01:01 was identified in the Chinese population. CONCLUSION: The PCR-SBT method for exon 3 of HLA-DRB1 from the present study was reliable and there were polymorphisms in exon 3 of HLA-DRB1.

[Cloning and expression of MHC class I chain-related gene A in E. coli].

In order to construct prokaryotic expression system of MHC classI chain-related gene A (mica) and purify MICA protein, RNAs were extracted from the peripheral blood samples and mica cDNA fragments were obtained by RT-PCR method. The cDNA for mica was ligated with cloning vector by TOPO method. The recombinant cloning vector and prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct pET-28a-MICA recombinant expression vector, then the pET-28a-MICA vector was transformed and expressed in E. coli BL21 DE3. The recombinant protein was purified by Ni-NTA Spin method. The results showed that the recombinant MICA protein expressed with soluble form in host with pET-28a-MICA vector after IPTG induction. The recombinant target protein was obtained by Ni-NTA spin purification. In conclusion, this study has constructed prokaryotic expression system of mica gene and has purified MICA protein which would help to explore the interaction between MICA and transplantation immunology.

[Molecular basis of a new O61 allele in ABO blood group].

Objective of this study was to explore the molecular basis of a new O61 allele in ABO blood group. The ABO group antigens on red cells of the blood samples were identified by monoclonal antibodies and the ABO antibody in serum was detected by the standard A, B, O red cells. The coding region sequences of exon 5 to exon 7 in ABO gene were amplified by polymerase chain reaction (PCR) and the amplification products were purified with double enzyme digestion and directly sequenced for exon 6 and 7. The diploid of the individual with B phenotype was separated into its haploid components with a haplotype specific extraction method. The exons 6 to 7 of the two single ABO haplotypes were then amplified and sequenced separately. The results indicated that 3 samples had mutation at 743 site in total 417 individuals, in which 2 individuals were with O phenotype and 1 individual was with B phenotype. The DNA sequencing of exon 6 and 7 in 2 samples with O phenotype showed 261G deletion and 743G/C heterozygotes. The DNA sequencing of exon 6 and 7 in the sample with B phenotype showed 261G/deletion and 297A/G, 526C/G, 743G/C, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A heterozygotes. After separating of the 2 single strands in the B sample with haplotype specific extraction, an O and B101 allele were identified after sequencing. The novel allele was submitted to the Blood Group Antigen Gene Mutation database and is named as O61. It is concluded that 743G>C is a novel mutation in exon 7 of ABO and a novel O61 allele with 743G>C has been identified.

[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.