Relationship between expression of chemokine receptor 2 and basic fibroblast growth factor in aqueous humor and prognosis of trabeculectomy in patients with acute primary angle-closure glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the relationship between the levels of chemokine receptor 2(CXCR2)and basic fibroblast growth factor(bFGF)in aqueous humor and the prognosis of trabeculectomy in patients with acute primary angle-closure glaucoma(APACG).METHODS: A total of 80 cases(80 eyes)APACG patients who underwent trabeculectomy in our hospital from June 2020 to January 2022 were collected in the case group. According to the postoperative efficacy, they were grouped into a success group of 60 cases(60 eyes)and a failure group of 20 cases(20 eyes). Another 86 cataract patients(86 eyes)who underwent phacoemulsification with normal intraocular pressure in our hospital during the same period were included in the control group. Enzyme linked immunosorbent assay was applied to detect the levels of CXCR2 and bFGF in aqueous humor. ROC curve was applied to analyze the value of predicting trabeculectomy failure in APACG patients by the levels of CXCR2 and bFGF in aqueous humor. Furthermore, multivariate Logistic regression was applied to analyze the influencing factors of trabeculectomy failure in APACG patients.RESULTS: The levels of CXCR2 and bFGF in the aqueous humor of the case group were significantly higher than those of the control group(P<0.05). The levels of CXCR2 and bFGF in the aqueous humor of the failed group and the proportion of patients with postoperative shallow anterior chamber were significantly higher than those of the successful group(P<0.05). The AUC for predicting trabeculectomy failure in APACG patients using CXCR2 and bFGF levels alone and in combination was 0.885, 0.883 and 0.953, respectively. CXCR2 and bFGF were independent risk factors for trabeculectomy failure in APACG patients(P<0.05).CONCLUSION: The levels of CXCR2 and bFGF in the aqueous humor of APACG patients are obviously elevated, and both are risk factors for trabeculectomy failure.

A study of HLA-DPA1 and DPB1 matching status for unrelated donor-recipient pairs matched at allele level for HLA-A, -B, -C, -DRB1 and -DQB1 loci / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the status of HLA-DPA1 and DPB1 matching for unrelated donor-recipient pairs matched at high-resolution allele level for HLA-A, B, C, DRB1 and DQB1 loci.</p><p><b>METHODS</b>A total of 76 unrelated donor-recipient pairs matching at allele level for HLA-A, B, C, DRB1 and DQB1 loci were subjected to HLA-DPA1 and DPB1 sequence-based typing (SBT). HLA-DPA1and DPB1 matching status at high-resolution allelic level was also analyzed.</p><p><b>RESULTS</b>The allelic identity ratio for single HLA-DPA1 and DPB1 were 17.1% and 9.2%, respectively. HLA-DPA1 and DPB1 allelic identity ratio were both very low. The majority of unrelated donor-recipient pairs (73.7%) had an incompatibility at 1 HLA-DPA1 allele, 9.2% of pairs had an incompatibility at 2 DPA1 alleles. As for the high-polymorphic HLA-DPB1 gene, 57.9% of studied donor-recipient pairs had an incompatibility at 1 HLA-DPB1 allele, almost 1/3 (32.9%) of them were completely incompatible. When HLA-DPA1 and DPB1 genes were analyzed together, the donor-recipient pairs matched at 2/4 was the most common (51.4%), 4/4 allelic complete matched pairs accounted for 5.6%, and 0/4 matched pairs accounted for 8.3%.</p><p><b>CONCLUSION</b>Our results indicated that the ratio of HLA-DPA1 and DPB1 complete match in the unrelated donor-recipient pairs matching at allelic level for HLA-A, B, C, DRB1 and DQB1 loci were very low. The effect of HLA-DPA1 and DPB1 matching status on clinical unrelated stem cell transplantation still needs to be elucidated.</p>

Establishment of a large-scale bi-directional sequencing and genotyping platform for MICA gene exons 2 to 4 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4, and to analyze single nucleotide polymorphisms(SNP) of the region.</p><p><b>METHODS</b>Primers for particular alleles of MICA gene exons 2 to 5 were designed. Optimal conditions for PCR amplification and sequencing reaction were explored. A commercialized one-way sequencing kit for MICA allele was used as a parallel control. Four samples carrying a MICA *010 allele were subjected to cloning and haplotype sequencing.</p><p><b>RESULTS</b>Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method. Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population. Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA*065 and MICA*066 by the WHO Nomenclature Committee. A novel SNP in MICA gene intron 3 was discovered, with allelic sequence submitted to GenBank and IMGT/HLA database.</p><p><b>CONCLUSION</b>The bi-directional sequencing genotyping platform may be applied for large-scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.</p>

Development of a high-throughput sequence-based typing assay for human leukocyte antigen loci / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a reliable assay for simultaneous sequence-based typing (SBT) of HLA-DPA1 and HLA-DPB1, and to apply it for the study of allelic polymorphisms in southern Chinese Han population.</p><p><b>METHODS</b>Based on full-length HLA-DPA1 and HLA-DPB1 allelic sequences, locus-specific PCR primers were designed and applied to amplify the target sequence encompassing the entire exon 2 of HLA-DPA1 and HLA-DPB1. PCR products were purified with magnetic beads, and run through an ABI 3730 DNA sequencer. Genotypes were assigned with an Assign 3.5 SBT software.</p><p><b>RESULTS</b>The target sequences of HLA-DPA1 and HLA-DPB1 were both amplified with the PCR procedure. Little background and noise was observed in the derived sequences. Among 176 non-related healthy individuals, 4 HLA-DPA1 alleles with the frequencies of DPA1*02:02 (0.589) > DPA1*01:03 (0.284) > DPA1*02:01 (0.096) > DPA1*04:01 (0.031) were identified. In addition, 14 HLA-DPB1 alleles, including 4 common alleles (with a frequency of more than 5%, namely DPB1*05:01, DPB1*02:01, DPB1*04:01 and DPB1*02:02), 7 alleles with a frequency ranging from 1%-5% and 3 alleles with a frequency of less than 1% were identified. The results of HLA-DPB1 genotyping were all in accordance with the typing results derived from an Atria AlleleSEQR HLA-DPB1 kit.</p><p><b>CONCLUSION</b>A reliable technique has been established for simultaneous genotyping of HLA-DPA1 and HLA-DPB1, which may have a broad application in population and disease association studies.</p>

Investigation and analysis of a common allele HLA-C*08:22 frequency in the Chinese southern Han population / 中国实验血液学杂志

This study was purposed to investigated and analyze the allelic frequency of a common allele HLA-C*08:22 in the southern Chinese Han population. A total of 32 samples with the C*08:01:01/08:22 ambiguous results previously identified in 163 unrelated southern Chinese Han population by routine sequencing based typing (SBT) at exons 2 - 4 of HLA-C gene were subjected to HLA-C SBT at exons 5 and 6 using our in-house method. Forty C*08:01:01-positive unrelated donor/recipient pair identified before the C*08:22 allele were officially nomenclatured and released by the World Health Organization (WHO) Nomenclature Committee for Factors of HLA System, were re-sequenced at exons 2 - 6 of HLA-C gene by our in-house SBT method. The allele assignment was accomplished with the Assign 3.5 SBT software. The results showed that three samples were identified as C*08:22-positive in the 32 samples with C*08:01:01/08:22 ambiguous results, the allele frequency of C*08:22 was 0.92% in the southern Chinese Han population. Retrospective analysis indicated that 2 donor/recipient pairs previously identified as C*08:01:01-positive were actually C*08:22-positive in the 40 tested donor/recipient pairs. It is concluded that the novel C*08:22 allele is the common allele in southern Chinese Han population, it can't be considered as rare allele and is ruled out for the samples with C*08:01:01/08:22 ambiguous results.

Cloning and sequencing analysis of a novel allele HLA-A*02:251 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel human leukocyte antigen (HLA) allele A*02:251 and analyze the sequences in Chinese population.</p><p><b>METHODS</b>Routine HLA-A, -B, -DRB1 high resolution genotyping for healthy Chinese donors and patients was performed with polymerase chain reaction-sequence based typing. An unknown HLA-A allele was initially detected by HLA typing in the healthy donor. Genomic DNA of the HLA-A locus in the proband was amplified, the amplified product was cloned by PMD18-T to split the two alleles, and selected clones were sequenced.</p><p><b>RESULTS</b>The sequencing results showed that a normal A*02:06:01 and a novel A*02:251 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (HM245348). Nucleotide sequence alignments with HLA-A allele from the IMGT/HLA Sequence Database showed that the novel A*02 variant allele differed from the closest allele A*02:01:01:01 by nt 383 G to C (codon 128 GAG to GAC) in exon 3, which resulted in one amino acid substitution of Glu to Asp. The HLA-A, B, C and DQB1 alleles of the healthy donor did not match with that of the patient.</p><p><b>CONCLUSION</b>This novel allele is officially designated as HLA-A*02:251 by World Health Organization(WHO) Nomenclature Committee (Submission ID HWS10010755). The sequence of HLA-A locus in exon 3 is confirmed to be polymorphic in Chinese population.</p>

Study on HLA nucleotide sequence matching in epitope positions among recipient-donor pairs for allogenic hematopoietic stem-cell transplantation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the human leukocyte antigens(HLA)-A, -B, -Cw, -DRB1 and DQB1 nucleotide sequences between patients waiting for allogenic hematopoietic stem-cell transplantation (HSCT) and donors in Chinese population, and to establish strategy for maximizing optimal donor selection.</p><p><b>METHODS</b>HLA high-resolution typing in a total of 537 recipient-donor pairs was determined by sequence based typing (SBT) method. The nucleotide BLAST tool was used to compare the nucleotide sequences among recipient-donor pairs.</p><p><b>RESULTS</b>Only 16.20% (88/537) of recipient-donor pairs were found to fully match for nucleotide sequences of all HLA-A,-B,-Cw, -DRB1 and -DQB1 loci. Mismatch rate in single locus were 8.38% in HLA-A, 0.74% in HLA-B, 12.29% in HLA-C, 2.42% in HLA-DRB1, and 2.79% in HLA-DQB1, respectively. Mismatch rate in two or multiple HLA loci was 42.65%. Nonpermissive allele mismatch combinations (A 02:01-A 02:06, A 02:06-A 02:07, Cw 03:04-Cw 15:02, Cw 03:03-Cw 04:01, Cw 03:04-Cw 14:02, Cw 03:03-Cw 08:01, DRB1 04:03:01-DRB1 04:05) were detected in single mismatch HLA locus of recipient-donor pairs, mismatches of B 07:05:01-B 07:06, Cw 07:01:01-Cw 07:06 combinations outside of epitope positions were detected in two recipient-donor pairs.</p><p><b>CONCLUSION</b>Our data suggested that attention should be paid in comparing nucleotide sequences between recipient and donor, and in distinguishing nucleotide sequence mismatches within and outside of the epitope positions. These results could serve as guidelines for donor selection.</p>

Genetic polymorphism of Chinese Zhuang population at HLA-Cw locus by sequence based typing / 中国实验血液学杂志

Thirst study was purposed to explore the genetic polymorphism of Chinese Zhuang population at HLA-Cw locus by sequence based typing (SBT). A total of 150 unrelated blood samples from Chinese Zhuang population were subjected to sequencing at exon 2, 3 and 4 of HLA-Cw gene in both directions by using SBT technique established by our laboratory. The purified products of sequencing reaction were run by means of electrophoresis on the ABI 3730 DNA Sequencer and the assignment of HLA-Cw genotype was accomplished by using the Assign 3.5 software. The consensus sequence at exon 2, 3 and 4 of HLA-Cw gene for each sample was imported into the Assign 3.5 software. The results showed that 33.33% of tested samples could obtain an unique genotype, genotype in 63.33% of tested samples with ambiguous results could be assigned by ruling out the rare alleles according to the NMDP Rare Allele List File; however, the final genotype in rest 3.33% of the detected samples could be defined when subjected to further confirmatory testing by PCR-SSP. In this detection 16 HLA-Cw alleles were identified, the common alleles with a frequency of > 10% were Cw*0304 > Cw*0102 > Cw*0801 > Cw*0702. The value for gene diversity (GD) was 0.9297, The frequency for Cw*01, 03, 07, 08, 12, 14 (Cw 1 allele group) and Cw*02, 04, 05, 06, 15, 16, 17, 18 (Cw 2 allele group) was 0.8967 and 0.1032, respectively, which indicated that the Cw 1 allele group is the dominant ligand for KIR in Chinese Zhuang population. 51 genotypes were determined and the distribution of genotype frequency was in line with Hardy-Weinberg principle. It is concluded that the obtained HLA-Cw allele frequency and its distribution characteristics of Chinese Zhuang population can provide valuable data in the studies of anthropology and the association of HLA-Cw with disease.

Demographic characteristics of patients with leukemia waiting for stem cell transplantation in Chinese Marrow Donor Program during 2000 to 2006 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the distributive characteristics for leukemia and to provide scientific reference for its prevention and intervention.</p><p><b>METHODS</b>Microsoft SQL 2005 databases was used to make a mathematical analysis of 3708 patients with leukemia in Chinese Marrow Donor Program (CMDP) from 2000 to 2006. The distributive characteristics were calculated by sex, age and area of patients with leukemia and then compared by constituent ratio and relative ratio statistics method.</p><p><b>RESULTS</b>A total of 3708 cases of leukemia were registered for waiting donor during the period 2000-2006 in CMDP, the age of patients were from 7 months to 69 years, the median age of diagnosis was 24.5 years, standard deviation was 6.7-years-old; males suffered more than females, and the ratio was 1.95: 1 (2451/1257). There were 1202 patients with acute lymphoblastic leukemia (ALL), 1066 with acute myeloid leukemia (AML), 1435 with chronic myeloid leukemia (CML), 5 with chronic lymphoblastic leukemia (CLL), CML was the most common patients. The distributive of 3708 patients with leukemia peak was from 15 to 30 years age group, 542 patients were at the age of 15 years, 559 patients were at the age group above 20 years, 514 patients were at the age above 25, 522 patients were at the age over 30-years-old. ALL patients were accounted for 49.36% (613/1242), AML patients accounted for 27.78% (245/1242), CML patients accounted for 22.78% (283/1242), CLL patients accounted for 0.08% (1/1242) in the age group of under 20 years (childhood group). All subjects were mainly in childhood patients with leukemia; The distributive of patients with leukemia in 30 areas were different, leukemia patients were not registered in one area, 494 patients were at the highest peak, 101 patients were in the median.</p><p><b>CONCLUSION</b>The majority of leukemia patients for waiting stem cell transplantation were registered among children and the adolescents groups, males were suffered more than the females. For children, the major type of leukemia was ALL, being necessary to pay more attention to the education of health, and the precaution of leukemia. The distributive of patients with leukemia for waiting stem cell transplantation was different in 30 areas, and the peak region of leukemia should be in Jiangsu, Guangdong, Shangdong, and Zhejiang provinces.</p>

Analysis of HLA haplotype frequency and linkage disequilibrium in patients with acute lymphoblastic leukemia from Northern Chinese Han / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the difference between the frequencies of HLA-A-B, B-DRB1 and A-B-DRB1 haplotype, as well as their linkage disequilibrium pattern in patients with acute lymphoblastic leukemia(ALL) and healthy controls from Northern Chinese Han.</p><p><b>METHODS</b>The frequencies of HLA-A-B, B-DRB1, A-B-DR haplotypes and linkage disequilibrium were estimated by Expectation Maximization method based on the genotypes of 643 patients with ALL and 2 0359 unrelated healthy donors, and the statistical significance between the two groups were estimated by chi-square test. Linkage disequilibrium was analyzed with population genetic methods.</p><p><b>RESULTS</b>The most common HLA-A-B, B-DRB1, and A-B-DR haplotypes were A30-B13, A2-B46, A33-B58, B13-DR7, B46-DR9, B52-DR15, B58-DR17, A30-B13-DR7, A33-B58-DR17 and A1-B37-DR10 in both groups. The frequencies of A30-B13, A2-B46, A33-B44, B13-DR7, A30-B13-DR7 and A2-B46-DR9 haplotypes and linkage disequilibrium value were significantly decreased (P<0.05) in the patient group than that in the control group. On the other hand, the frequencies of A2-B52, A31-B61, A24- B8, B60-DR9, B27-DR4, B52-DR14, B44-DR17, B27-DR12 and A11-B27-DR12 haplotypes and linkage disequilibrium value were significantly increased (P<0.05) in the patient group than that in the control group.</p><p><b>CONCLUSION</b>There are some common and positive linkage disequilibrium haplotypes in both the ALL patients and the healthy donors in Northern Chinese Han. Interestingly, some haplotypes and their linkage disequilibrium patterns had significantly different distributions between the two groups. The study provided basic data for the relationship of ALL and HLA haplotype and for finding the HLA-A, B, DR matching donors.</p>

Analysis on haplotypes of five HLA loci in southern Chinese Han population by sequence-based typing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the polymorphism and haplotypes of HLA-A, B, Cw, DRB1 and DQB1 loci in Chinese Han population.</p><p><b>METHODS</b>A total of 186 unrelated healthy individuals from southern China were analyzed by sequence-based typing. Two-, three-, and five-locus haplotypes were estimated using the Expectation Maximization Algorithm. RESULTST: Twenty-eight alleles for the HLA-A locus, 49 HLA-B alleles, 24 HLA-C alleles, 29 HLA-DRB1 alleles and 20 HLA-DQB1 alleles were detected. The A*0207-B*4601(10.81%), A*3303-B*5801(6.14%), B*4601-DRB1*0901(6.22%), B*4001*-DRB1*0901(3.78%), DRB1*090-DQB1*0303 (12.16%) and DRB1*1202-DQB1*0301(8.38%), A*0207-B*4601-Cw*0102 (10.75%), A*3303-B*5801-Cw*0302 (5.14%), A*0207-B*4601-DR*0901(5.07%), A*3303-B*5801-DRB1*0301(2.96%), A*0207-B*4601-Cw*0102-DRB1*0901-DQB1*0303(4.87%) and A*1101-B*1301-Cw*0304-DRB1*1501-DQB1*0601(2.43%) were the most common haplotypes in the southern Chinese Han population.</p><p><b>CONCLUSION</b>The results have shown the characteristics of the five HLA loci haplotype distribution and provided more information in anthropology, disease association studies and transplantation.</p>

Establishment of an assay for cloning and sequencing the full-length HLA-Cw gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a reliable assay for cloning and sequencing the full-length HLA-Cw gene.</p><p><b>METHODS</b>In this study, a fragment of 4.5 kb full-length HLA-Cw gene was amplified using the self-designed PCR primer pair by long template PCR, purified PCR products was cloned into the pGEM-Teasy plasmid vector and the plasmid DNA isolated from positive clones was subjected to haplotype sequencing by both directions. A total of 12 samples having been previously-genotyped by PCR sequence-based-typing (PCR-SBT) were amplified by using the TaKaRa LA Taq and Stratagene Pfu polymerase, respectively. PCR products of full length HLA-Cw gene were subjected to cloning and sequencing and the obtained haplotype sequence were compared with the PCR-SBT results.</p><p><b>RESULTS</b>The specific target fragment of HLA-Cw gene could be amplified and the full-length HLA-Cw allele sequence covering from nucleotide position -962 in 5'untranslated region (5'-UTR) to nucleotide position 3576 in downstream area of 3'-UTR region could be obtained using our method. The results of cloning and sequencing analysis indicated that the Stratagene Pfu polymerase had better fidelity than the TaKaRa LA Taq polymerase in this experiment. By comparing the sequences of Cw*07020101 with Cw*010201, 11 SNPs as well as 2 insertions/deletions in nt-962--284 of 5'-UTR, and 11 SNPs as well as 1 insertion/deletion in nt3067-3576 downstream of 3'-UTR were identified.</p><p><b>CONCLUSION</b>Our results indicate that the technique for cloning and sequencing full-length HLA-Cw gene has been established, it has a broad application in full-length HLA-Cw gene polymorphism study and the regulation and expression of HLA-Cw gene.</p>

Identification of a novel allele HLA-DRB1*1218 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel human leukocyte antigen (HLA) allele by cloning and sequence-based typing in Chinese population, and analyzing the sequence of the introns 1 and 2.</p><p><b>METHODS</b>The routine HLA-A, -B, -DRB1 low resolution genotyping for stem cell donor from Guangdong province was performed with polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). An unknown HLA-DRB1 allele was initially detected by HLA typing. Genomic DNA of the proband was amplified by using HLA-DRB1 locus group-specific primer, the amplified product was cloned, sequenced, and compared to the closest DRB1*120201 allele and the closest intron sequence of the DRB1*030101 allele.</p><p><b>RESULTS</b>The sequencing results showed that a normal DRB1*080302 and a novel DRB1*1218 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (FJ481086). The novel allele had 1 nucleotide substitution of the closest matching allele HLA-DRB1*120201 at nt262(G-->C) in exon 2,resulting in an amino acid change from Glu(GAG)-->Gln (CAG) at codon 59.The intron 2 sequence is identical between the novel HLA-DRB1*1218 and DRB1*030101, but there are 12 nucleotides substitution in intron 1.</p><p><b>CONCLUSION</b>A novel HLA allele was confirmed by cloning and sequence-based typing in Chinese. It was officially designated as HLA-DRB1*1218 by WHO Nomenclature Committee.</p>

An analysis of the reason for HLA-C allele dropout in five samples by sequence-based typing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the possible reason for HLA-C allele dropout in routine sequence-based typing (SBT) and improve the accuracy of HLA-C SBT test.</p><p><b>METHODS</b>A total of 620 randomly selected samples from healthy voluntary blood donors in Shenzhen were typed at HLA-C locus by sequence-based typing using the AlleleSEQR HLA-C plus sequence-based typing kit. Samples with no full match result were subjected to cloning and haplotype sequencing of the full-length HLA-C gene. If no novel mutations were found, samples were then retyped, using our self-designed PCR primer pair and PCR conditions replacing the AlleleSEQR HLA-C PCR reagents in the PCR set-up procedure so as to analyze the potential reasons for causing abnormal SBT result.</p><p><b>RESULTS</b>In the 620 samples typed at HLA-C locus using the AlleleSEQR HLA-C SBT commercial kit, 5 samples with no full match result were identified. The closest genotype showed one nucleotide mismatch with many different allele groups at different nucleotide position. Based on the PCR-SBT nucleotide sequence, heterozygous nucleotides were determined only in exon 4, whereas the nucleotides in exon 2 and 3 were all homozygotes. The results showed that HLA-Cw*0706 allele dropout existed in all the 5 samples with abnormal SBT results initially identified by AlleleSEQR HLA-C SBT kit, no novel mutation was found.</p><p><b>CONCLUSION</b>The results indicate that the PCR primer pair incompatible with DNA template may result in allele dropout in HLA-C SBT test. Based on the characterization of HLA-C full-length, it is essential to develop HLA-C SBT kit suitable for Chinese population in the future.</p>

High through-put genomic DNA isolation technique and its application in HLA genotyping for samples from bone marrow donor program / 中国实验血液学杂志

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.

A total of 362 HLA different haplotypes and HLA recombination haplotypes based on analysis of their family pedigree in Chinese partial Han populations / 中国实验血液学杂志

This study was aimed to discover the novel HLA recombination haplotypes and investigate the distribution of haplotypes in Chinese Han population. Based on the HLA-A, B, DRB1 typing results of 179 family members, 791 haplotypes were assigned by the mode of inheritance. The results showed that a total of 4 novel recombinant haplotypes in HLA-DRB1 locus region were observed in 4 families, which ratio of paternal to maternal chromosomes was 3:1. The recombination ratio between HLA-DRB1 and HLA-A or B loci was 0.92% (4/433). There were a total of 362 kinds of HLA-A, -B, -DRB1 haplotypes to be confirmed in Chinese Han partial population. A33-B58-DR17, A2-B46-DR9, A30-B13-DR7, A11-B13-DR15, A11-B75-DR12 and A2-B46-DR14 were the most common haplotypes that was consistent with the distribution of HLA alleles in unrelated donors. There were A1-B63-DR12, A29-B46-DR15, A1-B61-DR10, A34-B35-DR9, A29-B54-DR4, A23-B13-DR16 and A34-B62-DR15 haplotypes and so on, which were rare haplotypes not yet reported in Chinese. It is concluded that the HLA-A-B-DRB1 haplotypes would be confirmed by analysis of their family pedigree. The results obtained in this study are basic data for study of Chinese anthropology, organ transplantation and disease correlation analysis.

Establishment of genotyping method for human platelet antigens of HPA-15 system by PCR-SSP / 中国实验血液学杂志

This study was aimed to establish the reliable genotyping method of human platelet antigens of HPA-15 system by PCR-SSP and to use this assay in the further HPA genotyping of volunteer platelet donors. 3 sequence-specific primers recommended by the 11th Platelet Genotyping and Serology Workshop on behalf of International Society of Blood Transfusion (ISBT) were synthesized. The concentration of each primer pair, the concentration of Mg(2+) and the PCR conditions were adjusted to optimize the conditions so that HPA-15 system could be specific amplified. The accuracy and reliability of the developed assay was evaluated and confirmed by typing the coded DNA samples provided by the 11th Platelet Genotyping and Serology Workshop. As a parallel control, a total of 50 volunteer platelet donors in Shenzhen were genotyped by both our assay and the G&T commercial kit at HPA-15 system. 10 coded samples distributed by the 11th Platelet Genotyping and Serology Workshop were genotyped by established PCR-SSP method. The results showed that a concordance rate of 100% was observed between the results obtained by established PCR-SSP method and the results provided by ISBT report. The HPA gene frequencies observed in 50 randomly-selected platelet donors in Shenzhen were 0.5100 and 0.4900 for HPA-15a and HPA-15b respectively. In conclusion, PCR-SSP assay established in our study provides a simple, rapid and accurate method for HPA-15 system genotyping, which assay is suitable for routine clinical HPA genotyping and shows a broad prospect in its further applications.

Analysis on expression and molecular basis for ABO glycosyltransferase with dual specificity / 中国实验血液学杂志

In order to elucidate the expression and molecular genetic background of ABO gene seven samples with ABO discrepancy further identified as bi-specific ABO gene were studied. All these samples were subjected to phenotyping by monoclonal and polyclonal antisera and were then genotyped by direct DNA sequencing and haplotype-sequencing at the exon 6 and 7 of ABO gene. As a result, six ABO dual-specific alleles were identified in Chinese population. An antigen expressed by these B (A) or Cis-AB individuals varied from very low level to the normal level, compared with common A blood group samples. In conclusion, molecular genetic backgrounds of two pairs out of four samples in all samples were the same, however, the ABO expression showed diverse.

Genetic polymorphism of 9 Y-STR loci with short fragment size alleles in unrelated male individuals from Zhuang ethnic group / 中国实验血液学杂志

The aim of this study was to investigate the genetic polymorphism of Y-chromosome specific short tandem repeat (Y-STR) loci in Zhuang ethnic group of China. Nine Y-STR loci were amplified by single multiplex and the PCR products were detected by using ABI Prism(TM) 3100 DNA Sequencer. The allele frequencies and haplotype frequencies at 9 Y-STR loci were determined in a total of 85 unrelated male individuals from Zhuang ethnic group of China. The results indicated that in the 85 unrelated male individuals, except for the DYS426 locus with a low GD value, the GD values for other 8 Y-STR loci ranged from 0.4387 to 0.8129. A total of 70 haplotypes at 9 Y-STR loci were found, the haplotype diversity was 0.9926. It is concluded that the haplotype polymorphism of 9 Y-STR loci are highly polymorphic in Zhuang ethnic group and also significantly different from our previous reported data of unrelated male individnals in southern Chinese Han population.

Application of 17 Y-chromosome specific STR loci in paternity testing / 中国实验血液学杂志

The purpose of this study was to explore the ability of discrimination of the AmpFlSTR Yfiler PCR amplification kit containing 17 Y-STR loci and the allelic mutation in the practice of paternity testing in Chinese population. 36 non-paternity father/son pairs and 84 confirmed father/son pairs, which had been previously genotyped by using Reliagene Y-PLEX 6 commercial kit and the "9 Y-STR multiplex with reduced-size amplicons" developed by our laboratory, were subjected to Y-STR genotyping at 17 loci using the AmpFlSTR Yfiler PCR amplification kit. 17 Y-STR loci were amplified in single multiplex and the PCR products were detected by using ABI Prism 3100 DNA Sequencer. The number of Y-STR exclusion for each non-paternity father/son pair and the mutation events for each confirmed father/son pair were calculated and the observed results were compared with our previous reported data determined by Reliagene Y-PLEX 6 kit and the "9 Y-STR multiplex with reduced-size amplicons". The results showed that out of 36 non-paternity father/son pairs subjected to Y-STR genotyping by using the AmpFlSTR Yfiler kit, one case with no Y-STR exclusion of paternity and 35 cases with more than 3 Y-STR exclusions for each father/son pair were observed. The percentage of cases with more than 3 Y-STR exclusions in all the tested non-paternity cases for Yfiler kit was 97.22% (35/36), which was more than that of Reliagene Y-PLEX 6 kit (92.11%, 35/38) and our "9 Y-STR multiplex with reduced-size amplicons" (91.67%, 33/36). Except for single father/son pair with no Y-STR exclusion, an average of 11.3 Y-STR exclusions was observed in other 35 non-paternity father/son pairs. In the 84 confirmed father/son pairs, 5 mutation events with a single unit repeat change at DYS437, DYS439, DYS635, DYS389II and DYS19, respectively, were identified using the Yfiler kit. The average mutation rate was estimated at 3.50 x 10(-3) per locus per generation. The cases with Y-STR mutation events in all tested confirmed father/son pairs for Yfiler system were 5.95% (5/84), which was significantly higher than that of Y-PLEX 6 (2.15%, 2/93) and "9 Y-STR multiplex with reduced-size amplicons" (no mutation events in the same 84 confirmed father/son pairs). It is concluded that the Yfiler kit which allowing simultaneous analysis of 17 Y-STR loci offers a high ability of discrimination for paternity testing, however, the Y-STR allelic mutation of the Yfiler system can not be neglected.