Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent / 中国输血杂志

【Objective】 To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i. e. Ion Torrent S5 and Illumina Miseq. 【Methods】 A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company; 336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. 【Results】 Ambiguous results were found in 357 out of 434 samples, accounting for 82.3% (357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8% (275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7% (82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing; HLA-DPB1*13∶01∶01 were detected in 25 (58.1%, 25/43) and HLA-DPB1*107∶01 in 18 (41.9%, 18/43). 【Conclusion】 There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1*13∶01∶01/107∶01 alleles.

Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular mechanism for an individual with Bweak subtype.@*METHODS@#Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein.@*RESULTS@#Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function.@*CONCLUSION@#The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.

Analysis of HLA allele-specific antibodies and Eplets in patients with platelet transfusion refractoriness / 中国输血杂志

【Objective】 To analyze the specificity and Eplets of HLA allele-specific antibody in patients with platelet transfusion refractoriness (PTR). 【Methods】 HLA-A and B genotypes were detected by PCR-SBT, and HLA-Ⅰ antibodies in patients′ serum were detected by Luminex single antigen beads coating method. IPD-IMGT/HLA Database was used to find the differential amino acids of allele-specific antibodies, and HLA Eplet database was used to analyze the registry Eplets. 【Results】 HLA allele-specific antibodies were found in 12 out of 82 patients with PTR.After sequence alignment, a total of 18 differential amino acids were found, such as 19E>19K, 166D>116E, 167G>167W and so on. Among these differential amino acids, 16 registry Eplets were retrieved such as 19E>19K, 95I>95L, 113YD>113HD and so on.The amino acid substitution of 166DG>166EW, 70Q>70H, 67S>67Y, 94I>94T, 82LR>82RG, and 211G>211A may form new Eplets that have not been registered.The antigens of A11, A24, B15, B27 and B38 can be further subdivided into HLA narrow specific antigens. 【Conclusion】 It was found that there were HLA allele-specific antibodies in patients with PTR, suggesting that high-resolution typing of HLA-A, B should be carried out for these patients and platelet donors in HLA compatible transfusion of PTR.

Construction and application of platelet virtual matching information system / 中国输血杂志

【Objective】 To construct a platelet digital matching information system (PMIS). 【Methods】 The framework of PMIS was designed and the main functions were developed. The information system was connected with the information modules of clinical application, laboratory testing, blood donation service, blood inventory and distribution. Further, the preliminary application of this system will be carried on in clinical. 【Results】 The PMIS had been successfully developed and 5048 blood donors with HLA and HPA genotypes were registered in the system. A total of 306 patients applied for matching and 16.5% of them received compatible platelet reports immediately from the inventory bloods, with the median waiting time of all matching as 3 days, which was significantly shorter than that of the manual method (3.8±3.1 days vs 5.4±5.4 days). 【Conclusion】 The developed PMIS has realized the whole process management of blood donors and patients, which is helpful to improve the platelet matching level.

Analysis of HLA-Ⅰ antibody specificity and estimation of antigen immunogenicity: 96 patients recieved genotype-matched platelet transfusions / 中国输血杂志

【Objective】 To analyze the specificity of HLA class-Ⅰ antibody in the patients received HLA-matched platelet transfusions and estimate the relative immunogenicity of HLA-Ⅰ antigens. 【Methods】 The samples from 96 patients who suffered from platelet transfusion refractorines(PTR) and applied for transfusion with genotype-matched platelet were collected. The specificity of HLA I antibody was detected by Luminex technique, and the antibody expression level was analyzed according to MFI. The mismatch rate of HLA antigen and relative immunogenicity of the population were estimated according to the allele frequency distribution of HLA-A and B loci as well as the yielding frequency of antibody. 【Results】 HLA-Ⅰ antibodies were detected in all 96 patients, with varied species of antibodies. The average positive yielding rates of antibodies corresponding to HLA-A, -B and -C magnetic bead coated antigens (97 in total) were 0.38, 0.47 and 0.28, respectively. Among the HLA-A and -B loci in the Zhejiang population, HLA-A2, A11, A24 and HLA-B60, B46, B58 were the antigens with higher frequency, and their relative immunogenicity was 0.403, 0.283, 0.342, and 0.100, 0.067, 0.178, respectively. 【Conclusion】 The specificity of HLA-Ⅰ antibodies in PTR patients is different, which confirms that the relative immunogenicity differs by HLA-A and -B antigens.

Correlation between the quantitative intensity of HLA-Ⅰ gene and its antibody and the clinical transfusion effect of matching platelets / 中国输血杂志

【Objective】 To explore the influence of anti-HLA-Ⅰ with different mean fluorescence intensity (MFI) on the efficacy of HLA-A and -B gene matching platelet transfusion, so as to provide scientific data for clinical platelet gene matching transfusion strategy. 【Methods】 A total of 81 PTR patients had applied for HLA-Ⅰgene matched platelets from the platelet gene database established by our laboratory, and 28 (MFI <5 000) of them needed further avoiding of partial donor-specific antibodies and they were enrolled as the research subjects. According to the platelet MFI value of HLA-Ⅰ antibody-targeting antigen, they were divided into negative transfusion group (MFI <500) (group A) and positive transfusion groups (MFI≥500) ; the latter were further divided into group B (500≤MFI <1 000), group C (1 000≤MFI <3 000) and group D (MFI≥3 000) according to MFI value. Corrected count increment (CCI) in platelet count was used to compare the platelet transfusion effect in 4 groups. 【Results】 Among 28 platelet recipients with MFI <5 000, 19(67.86%) patients successfully received 72 effective transfusions. The first CCI (×109/L) in groups A, B, C and D were 10.27±7.46, 7.58±4.75 (P>0.05), 17.36±7.63 (P>0.05) and -0.77±2.30 (P<0.05), respectively. There was no statistical difference among group A, B and C. 【Conclusion】 The application of HLA-Ⅰ gene matching platelets in PTR patients can adjust the MFI threshold(<2 000) appropriately according to the patient′s condition without compromising the platelet transfusion effect.

Analysis and prospect of platelet compatible transfusion / 中国输血杂志

Platelet compatible transfusion can effectively solve the immune mediated platelet transfusion refractoriness (PTR), save platelet resources and improve blood safety. This paper comments and prospects the compatibility modes of HLA, HPA and CD36, HLA antibody titer, antigen immunogenicity and the development of platelet compatible transfusion. The pattern of HLA compatible platelets involves the matching in the alleles, antigens and epitopes levels, respectively, as well as avoidance donor specificity antibody (DSA) method. While setting the mean fluorescence intensity (MFI) threshold of avoidance DSA needs to be explored when using the DSA prediction method. Allele specific HLA antibodies can be found in the patients with PTR. Therefore, the patients and donors should be genotyped for HLA-A, -B loci at high-resolution level in order to avoid allele specific HLA antibodies. The immunogenicity of various antigens or epitopes at HLA-A and -B loci are different. Selecting donor platelets with low antigen expression or low immunogenicity may be a way of HLA compatible platelets. As the probability and type of HPA antibody production are different in the various populations, the approaching of compatibility HPA involves allele matching and avoidance DSA. As to CD36, the compatibility mode mainly refers to avoidance DSA, which means blood donors with CD36 antigen type Ⅰdeficiency are preferentially selected, and then those with CD36 antigen type Ⅱ deficiency. In the future, more attention should be paid to the scale up of database capacity and update of the information construction. The time waiting for compatible platelets transfusion in clinical could be significantly shortened if the requiring and matching are only conducted within the inventory and candidate platelets.

Analysis of loss of heterozygosity at HLA loci in a patient with leukemia / 中华医学遗传学杂志

OBJECTIVE@#To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation.@*METHODS@#HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions.@*RESULTS@#After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample.@*CONCLUSION@#LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

Study of the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang / 中华医学遗传学杂志

OBJECTIVE@#To study the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang.@*METHODS@#Genomic DNA was extracted by using a magnetic bead method. The full sequence of the KIR3DL2 gene was amplified with four pairs by PCR primers. The coding regions of 208 unrelated ethnic Han Chinese blood donors were analyzed using a BigDye Terminator v3.1 Sequencing Kit. The genotypes were assigned based on the nucleotide polymorphism of the KIR3DL2 gene.@*RESULTS@#Among the 208 samples, 133 were KIR3DL2 heterozygotes and 75 were homozygotes. Forty six KIR3DL2 genotypes were detected. Respectively, 70, 33 and 23 individuals were found to have a KIR3DL2*00201/KIR3DL2*00201, KIR3DL2*00201/KIR3DL2*00701, and KIR3DL2*00201/KIR3DL2*01001 genotype. Twenty-two KIR3DL2 alleles were discovered, and the frequencies of KIR3DL2*00201, KIR3DL2*00701 and KIR3DL2*01001 were 57.45%, 13.46% and 9.13%, respectively.@*CONCLUSION@#The distribution of KIR3DL2 alleles among ethnic Han Chinese in Zhejiang has been determined and fits the criteria for genetic polymorphism.

Identification of a glycosyltransferase allele associated with Bw subtype and analysis of the protein structure / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for an individual with Bw subtype.@*METHODS@#Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed.@*RESULTS@#Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased.@*CONCLUSION@#The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.

Molecular characterization of a recombination allele of ABO blood group / 中华医学遗传学杂志

OBJECTIVE@#To analyze the molecular characteristics of a recombinant allele of the ABO blood group.@*METHODS@#The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing.@*RESULTS@#The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived.@*CONCLUSION@#An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.

HLAⅠ-matched transfusion for immune-mediated platelet transfusion refractoriness / 中国输血杂志

【Objective】 To establish the HLA-A, -B genotype-matched transfusion strategy for immune-mediated PTR patients based on donor HLA genotyping database, so as to improve the transfusion efficacy. 【Methods】 The serologic cross-match was used to screen immune PTR primarily. 35 PTR patients screened out were subjected to HLA-match. 24 patients were tested for HLA-A, -B genotyping and antibodies against platelet HLA classⅠ, and then received a total of 83 HLA-typed platelet transfusions, based on patient platelet genotype, donor specific antibody (DSA)(priority), and HLA-A, -B cross-reactive groups (CREGs) principle(lower priority). The other 11 patients received a total of 55 HLA-A/B-matched transfusions according to CREGs principle. The clinical information and transfusion outcome were followed up, and the corrected count increment (CCI) was calculated and statistically analyzed. 【Results】 A total of 453 ABO serological cross-matching tests were performed for 35 PTR patients, with 12.94 tests (453/35) per patient, an average of 4.21 (1908/453) donors per test and positive rate of 69.86% (1333/1908). 23 out 24(95.83%) patients, subjected to HLA class I antibody, were positive and each carried (44.37 ± 22.31) kinds of specific antibodies. According to the fluorescence intensity of the antibody in the patient′s serum, the antibody was strongly positive in 17(73.91%) cases, positive 20(86.96%) and weakly positive 23(100%). After 138 HLA-matched transfusions, the first mean CCI value was 14.08 ± 11.12 (23.95 ± 21.28 h), which was significant higher than 1 hour CCI (>7.5 effective) or 24 hours CCI (> 4.5 effective). The responses of DSA avoidance group (CCI of 1st =15.56±11.00)was significant higher than that of non-DSA avoidance group(CCI of 1st =11.86±12.00)(t=2.045, P<0.05). 49.28% of the patients had one or more non-immune factors during platelet transfusion. 【Conclusion】 The HLA-matched platelet transfusion is feasible to prevent and improve immune-mediated PTR. For patients with multiple blood transfusions and positive platelet antibodies, DSA avoidance and CREGs principle combined transfusion strategy can significantly improve the efficacy of blood transfusion and provide accurate platelet transfusion for the clinical.

Detection of anti-HLA and anti-MICA in convalescent plasma from individuals recovered from COVID-19 / 中国输血杂志

【Objective】 To analyze the positive rate of antibodies against human leukocyte antigen(HLA)and MHC class I chain-related gene A(MICA) in the convalescent plasma from individuals recovered from COVID-19. 【Methods】 HLA-Ⅰ, -Ⅱ and MICA antibodies were screened simultaneously by Luminex platform. The specificity of HLA-Ⅰ and -Ⅱ antibodies was identified by single antigen reagents.The positive rate of antibody in different groups were compared by Chi-square test. 【Results】 A total of 88 cases of convalescent plasma were collected, among which the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies were 18.19%, 19.32% and 10.23%, respectively, and 64 individuals (72.73%) were negative for HLA-Ⅰ and -Ⅱ antibodies. 95 blood donors were randomly selected as the control group, and the positive rate of HLA-Ⅰ, -Ⅱ and MICA antibodies were 8.42%, 13.68% and 10.53%, respectively, and 76 individuals(80.00%) were negative for HLA-Ⅰ and -Ⅱ antibodies. There were no significant difference in the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies between convalescent individuals and control group. The specificity of HLA antibody to epitopes was different in each convalescent individual with positive HLA antibodies, and most antibodies were targeted to the epitopes of multiple HLA alleles. 【Conclusion】 A certain proportion of HLA antibody was found in the convalescent plasma of individuals recovered from COVID-19. Therefore, HLA antibody screening is helpful to improve the safety of transfusion.

The effects of blood donors satisfaction survey based on computer-aided technology / 中国输血杂志

【Objective】 To evaluate the application of computer-aided personal interviewing (CAPI) and computer-assisted telephone interview (CATI) in the satisfaction survey among blood donors. 【Methods】 Voluntary blood donors from stationary and mobile blood collecting sites of Zhejiang Blood Center from Jan 2018 to Dec 2020 were surveyed according to the principle of random sampling. The satisfactory degree of totally 21 items involving pre-donation, in-donation, and post-donation were surveyed. The outcomes were categorized and subsequently analyzed by SPSS23.0, and the differences in demography and blood types between different years were analyzed. 【Results】 5 534 voluntary blood donors were investigated via CAPI and CATI, which contributed to the reflection of real needs of blood donors. The results showed that the comprehensive satisfactory degree of blood donors increased from 95.4% in 2018 to 95.8% in 2020. Significant difference has been found in the eight indexes in Zhejiang (P<0.05) as blood donation publicity, environment of blood donation, service and professional skills of healthcare physicians, skills of phlebotomist before donation, service of pre-donation testing physicians, service and skills of phlebotomist during donation. 【Conclusion】 The combination of CAPI and CATI methods used in the survey of blood donor satisfactory degree, which has presented credible results, is more objective, convenient and time saving, therefore worth popularizing.

Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.</p><p><b>METHODS</b>The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.</p><p><b>RESULTS</b>DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.</p><p><b>CONCLUSION</b>Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.</p>

Study of the molecular basis for an individual with Bel variant due to deletion of B glycosyltransferase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis of an individual with Bel variant of the ABO blood group.</p><p><b>METHODS</b>The ABO antigen and serum antibody of the individual were detected by serological method. All coding regions and flanking introns of the ABO gene were amplified with PCR and sequenced bidirectionally. The haplotypes of the individual were analyzed by cloning and sequencing. A three dimensional model of the mutant protein was constructed and analyzed.</p><p><b>RESULTS</b>The individual has expressed a very weak B antigen on its red blood cells by absorption and elution testing, which was identified as a Bel variant phenotype. The heterozygous sites in exon 6 (261del/G) and exon 7 (297A/G, 484del/G, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A) of the coding region of the ABO gene were identified by direct sequencing. Haplotype analysis showed that the individual has carried an O01 allele and a novel B allele. The sequence of the novel B allele was identical to B101 except for a del G at nucleotide position 484 (484delG), which was nominated as B120 by the Blood Group Antigen Gene Mutation Database (dbRBC NCBI). The 484delG mutation of the B allele has led to a reading frame shift and created a premature terminal codon for the glycosyltransferase (GT) enzyme. Prediction of the 3D structure suggested that the GT enzyme has become an incomplete protein only with its N-terminal region.</p><p><b>CONCLUSION</b>The 484delG mutation of the glycosyltransferase B gene has probably abolished or reduced the enzymatic activity and resulted in the Bel variant phenotype.</p>

Study of the molecular basis of an individual with Aw43 subtype of the ABO blood group / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis of an individual with A subtype of the ABO blood group.</p><p><b>METHODS</b>The ABO antigen and serum antibody of the proband and his parents and sister were detected by a serological method. The whole coding regions of the ABO gene were amplified by PCR and subjected to bidirectional sequencing.</p><p><b>RESULTS</b>Red blood cells of the proband showed mixed field agglutination with anti-A but did not agglutinate with anti-B, and his serum did not agglutinate with A and O cells but with B cells. The proband was identified as an Aw phenotype. Heterozygous status of 1A/G, 106G/T, 188A/G, 189C/T, 220C/T, 261G/del, 297A/G, 467C/T, 646A/T, and 681A/G of the coding region of the ABO gene were identified by directly sequencing of the proband. The serological characteristics and nucleotide sequences of the mother were similar to those of the proband. However, the ABO genotypes of his father and sister were B101/O02 and O02/O02. The proband therefore has carried an O02 allele and a novel allele. Compared with A102, the novel allele contains 1A>G, which resulted in translation-initiator code change and was nominated as Aw43 by dbRBC of NCBI.</p><p><b>CONCLUSION</b>An Aw43 subtype has been identified for the first time, which may be attributed to the 1A>G and 467C>T variants on the α1,3-N-acetyl-galactosaminyltransferase gene.</p>

Study of in vitro expression of human platelet ITGB3 gene nonsense mutation c.1476G>A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.</p><p><b>METHODS</b>An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.</p><p><b>RESULTS</b>The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.</p><p><b>CONCLUSION</b>The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.</p>

Establishment of a polymerase chain reaction sequencing based typing method for HLA-DPB1 exons 2 and 3 and investigation of their polymorphisms / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a polymerase chain reaction sequencing-based typing (PCR SBT) method for HLA-DPB1 exons 2 and 3, and to analyze their polymorphisms.</p><p><b>METHODS</b>Based on the sequences of HLA-DPB1 loci, locus-specific primers were designed and applied to amplify the target sequences encompassing the entire exons 2 and 3 of HLA-DPB1. The amplification products were digested by enzymes and directly sequenced in both directions. The genotype was assigned by Assign 3.5+ SBT software.</p><p><b>RESULTS</b>Specific target fragment was obtained with the PCR amplification, and good quality electropherogram was derived by direct sequencing. Among 242 individuals from Zhejiang Han population, 18 HLA-DPB1 alleles were detected. Alleles with a frequency of > 0.05 have included DPB1*05:01:01/135:01 (0.4112), DPB1*02:01:02 (0.1901), DPB1*04:01:01 (0.1136) and DPB1*02:02 (0.0620). A novel HLA-DPB1*168:01 allele has also been identified. Nine polymorphism sites were founded in the exon 3 region, which included a new SNP site 517 A>T.</p><p><b>CONCLUSION</b>The PCR-SBT method for exons 2 and 3 of HLA-DPB1 is reliable, which allowed detection of polymorphisms in exon 3 of the HLA-DPB1 gene.</p>

Molecular basis for an individual with rare p phenotype in P1Pk blood group system / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system.</p><p><b>METHODS</b>Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing.</p><p><b>RESULTS</b>The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position.</p><p><b>CONCLUSION</b>The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.</p>