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One nucleotide substitution in codon 189 of HLA‐C*01:02:01:01 results in a novel allele, HLA‐C*01:179. [ABSTRACT FROM AUTHOR]

Objective: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system., Methods: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDx TM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods., Results: A total of 139 alleles were detected, including HLA-DRB1 (45), DRB3 (7), DRB4 (5), DRB5 (7), DQA1 (17), DQB1 (21), DPA1 (10) and DPB1 (27). HLA-DRB1 *09:01(17.09%),15:01(10.72%); DRB3 *02:02(25.99%),03:01(10.18%); DRB4 *01:03(36.46%); DRB5 *01:01(15.42%); DQA1 *01:02(20.01%),03:02(17.19%); DQB1 *03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1 *02:02(54.45%), 01:03(31.18%) and DPB1 *05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ 2 =12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1 *04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System., Conclusion: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Background: The null phenotype in P1PK blood group, known as "p," is extremely rare in the whole world. Individuals of p phenotype spontaneously form anti-PP1P K isoantibody. Here, we report a case of p phenotype with naturally occurring anti-PP1P K isoantibodies in a Chinese individual., Study Design and Methods: Serology tests, containing alloantibodies screening and identification, were conducted to demonstrate the phenotype in P1PK blood group. The genotype of A4GALT gene was identified by haplotypes separation and sequencing., Results: The serological assay demonstrated the p phenotype of the proband, presenting with 1:64 titer of anti-PP1P K . The sequencing data revealed a compound heterozygote consisting of A4GALT*P1.01 with c.343A>T and a novel allele based on A4GALT*01N.05 with an addition polymorphism c.100G>A. The sequence of the novel allele has been submitted to GenBank and the accession number OM912503 was assigned., Conclusion: Our study demonstrates a case of naturally occurring anti-PP1Pk in a Chinese individual with p phenotype, which is based on compound heterozygosity including one novel allele. As the proband is a young lady, monitoring the titer of anti-PP1P K and early initiation of medical intervention are essential after her pregnancy., (© 2022 AABB.)

The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), an actin nucleation-promoting factor, is present in the nucleus where it regulates gene transcription and maintains nuclear organization. Here, we show that WASH interacts with core non-homologous end-joining (NHEJ) factors including Ku70/Ku80 and DNA-PKcs, and Ku70/Ku80 is involved in the recruitment of WASH to the sites of DNA double-stranded break (DSB). WASH depletion leads to increased cell sensitivity and impaired DNA repair capacity in response to etoposide-induced DSBs and reduces NHEJ efficiency. Mechanistically, we show that loss of WASH inhibits the phosphorylation of DNA-PKcs, H2AX, and KAP1 after DSB induction and reduces chromatin relaxation and the recruitment of several downstream NHEJ factors to DSBs. Moreover, WASH role in DSB repair depends on its conserved C-terminal VCA domain and Arp2/3 activation. Our findings reveal a function and mechanistic insight for WASH in DNA DSB repair by the NHEJ pathway., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus., (© 2015. Published by The Company of Biologists Ltd.)

Objective: 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., Methods: 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., Results: 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%., Conclusion: 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.

Objective: To explore the cell signal transduction pathway of calcium-sensing receptor (CaSR) mediated hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs)., Methods: The expressions of proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated protein kinase 1, 2 (ERK1, 2) were analyzed by Western blot. Cell proliferation was tested by a 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell cycle and proliferation index (PI) were analyzed by flow cytometry., Results: Hypoxia significantly increased the expression of PCNA (0.528 ± 0.028), p-ERK1, 2 (1.12 ± 0.05, 0.91 ± 0.06), BrdU incorporation (143.3 ± 4.2) and cell proliferation index (12.5 ± 0.9) (all P < 0.05, versus control group, 0.243 ± 0.025, 0.47 ± 0.03, 0.40 ± 0.03, 100.0 ± 5.4, 7.5 ± 1.2). Gadolinium chloride (GdCl3, a CaSR agonist) amplified the effect of hypoxia (0.770 ± 0.039, 1.50 ± 0.06, 1.61 ± 0.05, 187.4 ± 3.9, 19.8 ± 0.6, all P < 0.05). PD98059 (a MEK1 inhibitor) decreased the up-regulation of PCNA expression, BrdU incorporation and the increase of cell proliferation index induced by hypoxia and GdCl3 in PASMCs (0.441 ± 0.020, 0.71 ± 0.07, 0.72 ± 0.06, 115.5 ± 4.0, 9.3 ± 1.1, all P < 0.05)., Conclusion: Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through ERK1, 2 pathways.

Objective: 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., Methods: 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., Results: 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., Conclusion: The bi-directional sequencing genotyping platform may be applied for large-scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

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.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

Objective: To identify a novel human leukocyte antigen (HLA) allele A*02:251 and analyze the sequences in Chinese population., Methods: 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., Results: 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., Conclusion: 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.

Objective: To analyze the human leukocyte antigen complex class I (-A, -B & -C) and class II (-DRB1 & -DQB1) linked haplotypes of Guangdong Han nationality and to study the recombination events of five classical loci in the inheritance of HLA haplotypes., Methods: A total of 939 peripheral blood samples were collected from 198 families in Guangdong Han nationality who came to our center for HLA typing from 2000 August to 2009 December. HLA-(A, B & DRB1) and HLA-(C & DQB1) alleles were typed by low-resolution polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO) and PCR-sequence specific primers (PCR-SSP) methods respectively. Then the recombination sites were analyzed by familial study. The samples of 52 individuals from the families with exchange recombination were analyzed by the sequence-based typing (SBT) to judge whether the recombination was interallelic or interlocus exchange., Results: Among 543 offspring individuals of 198 families in Guangdong Han nationality, 9 individuals with HLA-A-C-B-DRB1-DQB1 linked haplotypes had a recombination rate of 1.657%. Among 9 HLA haplotypes recombined families, 3 of them were found to have a crossover between HLA-A and -Cw loci and 6 of them a crossover between HLA-B and -DRB1 loci. Four of these recombination events occurred in the most common haplotypes A*3303-Cw*0302-B*5801-DRB1*0301-DQB1*0201 of Guangdong Han nationality. Among 9 cases of recombination, 5 of them were formed by a crossover between maternal chromosomes and 4 cases a crossover between paternal chromosomes. Three individuals with an exchange between A/Cw loci were all females. Among 6 cases with an exchange between B/DRB1 loci, 5 of them were males and 1 case was female., Conclusion: During the inheritance, recombination of HLA linked haplotype mainly occurred between A/Cw loci and B/DRB1 loci, the recombination is related to the haplotype-specificity and sex-specificity.

In the present study, a high-resolute method for cloning and sequencing genomic full-length HLA-A and -B using 20 Chinese Han individuals was established. We detected 10 HLA-A allele sequences 4.2 kb in length and 6 HLA-B allele sequences 3.7 kb in length, and the sequences included all exons, all introns, 5'promoter, and 3'UTR of the two genes. All sixteen sequences have been submitted to GenBank and IMGT/HLA database. A*1153 is a novel allele, and the introns of B*151101 are firstly reported here. The 5'promoter and 3'UTR sequences of 5 HLA-A alleles and 2 HLA-B alleles are also firstly disclosed, and all other alleles have extended the genomic full length sequences released in IMGT/HLA database. The polymorphic structures of upper 5'promoter and downstream 3'UTR, which were uncovered in IMGT/HLA database, are firstly depicted in Chinese Han individuals. Twenty-six single nucleotide polymorphisms (SNPs) and one 3 bp-insertion/deletion (Indel) were located in the upper 5'promoter and 14 SNPs were located in the 3'UTR of HLA-A. In addition, five SNPs and one 1 bp-indel were located in the upper 5'promoter and 5 SNPs were located in the 3'UTR of HLA-B. Through analyzing the phylogenetic trees of 5'promoter, exons and 3'UTR of the two genes, we found that the evolution history of regulatory regions and exons is different between the two genes. The regulatory regions are tightly linked with exons in most of HLA-A alleles excluding A*24020101. On the contrary, recombinant events may occur frequently between regulatory regions and exons in most HLA-B alleles.

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.

To analyze the molecular genetic polymorphism of full-length HLA-Cw gene, a total of 28 samples with known genotypes from Chinese Han population were amplified by long-range PCR using high-fidelity Pfu polymerase. A fragment 4.5 kb in length of HLA-Cw gene was subjected to cloning and haplotype sequencing. The single nucleotide polymorphisms (SNPs) in all segments of the whole region of HLA-Cw gene were analyzed. As a result, we detected 22 different HLA-Cw alleles in 28 samples, all of which were submitted to GenBank and the IMGT/HLA Database. Among the 22 HLA-Cw alleles, the intronic sequences of Cw*030301, Cw*0706 and Cw*140201 were firstly elucidated. The novel intronic sequence and the SNPs information may help to design allele-specific primers for accurate sequence-based typing (SBT) and to avoid allele dropout events in SBT test. We aligned all the diploid sequences using ClustalX program and imported them into Dnasp4.0 to calculate polymorphism in all coding- and non-coding regions. We found 244 SNPs and 10 insertion/deletions (Indels). According to the analysis of polymorphism level, phylogenetic trees and frequency spectrum, we proposed that the evolution of intron 4 and exon 5 was under balancing selection. Selection on these segments indicated that they may be functionally important in evolution of HLA-Cw gene. The full-length sequences obtained and related SNPs information can be used as resources of markers for high-resolution typing, complex diseases association studies and human evolution.

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.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

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.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

Objective: To establish a reliable assay for cloning and sequencing the full-length HLA-Cw gene., Methods: 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., Results: 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., Conclusion: 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.

Objective: To analyze the polymorphism and haplotypes of HLA-A, B, Cw, DRB1 and DQB1 loci in Chinese Han population., Methods: 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., Conclusion: The results have shown the characteristics of the five HLA loci haplotype distribution and provided more information in anthropology, disease association studies and transplantation.

Objective: 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., Methods: 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., Results: 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., Conclusion: 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.

Objective: To explore the distributive characteristics for leukemia and to provide scientific reference for its prevention and intervention., Methods: 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., Results: 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., Conclusion: 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.

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.

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.

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.

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.

To study the genetic polymorphisms of Y-chromosome-specific STR loci in Chinese of different surnames, 9 Y-STR loci were amplified by single fluorescent multiplex PCR and the PCR products were detected by using ABI PrismTM 3100 DNA Sequencer. Samples were randomly-selected from male blood donors in ShenZhen. These individuals, who were otherwise unrelated, had the most common surnames among Chinese as their surnames, namely Li, Wang or Zhang. There were 139 subjects with surname Li, 118 with surname Wang and 119 with the surname Zhang. In the Li population, a total of 126 haplotypes was found and 118 of them were unique, with a haplotype diversity 0.9974. In the Wang population, a total of 105 haplotypes was found and 94 of them were unique, with a haplotype diversity of 0.9953. In the Zhang population, a total of 101 haplotypes was found and 88 of them were unique, with a haplotype diversity of 0.9964. Our results indicated that the genetic polymorphisms of Y-STR haplotypes at these 9 loci in unrelated male individuals with Chinese surnames of Li, Wang or Zhang are highly polymorphic and showed no significant differences with our previous data of haplotype polymorphisms in unrelated male individuals from the Chinese ethnic Han population.