Objective:To study the genetic polymorphisms of short-tandem repeats (STR) for the D13S317 locus among an ethnic Han Chinese population and verify a novel tri-allelic pattern identified for the locus. Methods:A total of 378 paternity test cases from Guangdong Forensic Authentication Institute from October 17, 2017 to December 28, 2017 were selected as the study subjects. A GlobalFiler? Express kit was used for the STR genotyping. Samples suspected for having a novel tri-allelic pattern were verified with a PowerPlex ? 21 kit. Potential variant of the primer-binding region and flanking sequences underlying the tri-allelic pattern was excluded by molecular cloning and sequencing. Results:Six alleles were detected for the D13S317 locus, with the characteristic distribution frequencies being 8 (29.1%), 9 (13.1%), 10 (15.21%), 11 (24.21%), 12 (13.89%) and 13 (3.44%), respectively. In one of the families, the D13S317 locus of the proband was suspected to harbor a triband allele (8, 9, 10). A re-test has confirmed the result of initial test. Molecular cloning and sequencing analysis of the D13S317 locus in the proband and his daughter has failed to find allelic variants in the primer-binding region and flanking sequence, which has confirmed the novel tri-allelic pattern for the locus. Conclusion:A novel type 2 tri-allelic pattern (8, 9, 10) at the D13S317 locus has been identified among the ethnic Han Chinese population. The pattern has not been transmitted to the female offspring, and has been included in the international STRBase database for the first time.

Objective:To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree.Methods:A female patient with acute myeloid leukemia who had visited Liuzhou People′s Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene.Results:The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35: 01, 40: 02 and HLA-B*35: 01, 40: 01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35: 01 at exon 4. NGS results showed that the HLA-B*35: 01 had a 9 bp deletion in the intron 5. The patient′s husband was HLA-B*40: 01, 58: 01, which was normal. Conclusion:The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.

Objective:To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population.Methods:Using PCR - SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals, and then using the method of high fidelity and long-fragment PCR - SBT to amplified, sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals.Results:We successfully amplify the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population. 209 KIR2DS4 positive individuals were detected, and the positive rate is 94.1%. By sequence-based typing, we identified 12 genotypes and 7 alleles of KIR2DS4. The 6 known alleles and their detection frequency is as follows: KIR2DS4 * 00101/011 (180, 81.1%), KIR2DS4 * 010 (53, 23.9%), KIR2DS4 * 004 (34, 15.3%), KIR2DS4 * 003 (15 and 6.8%), KIR2DS4 * 006 (2, 0.9%) and KIR2DS4 * 015 (1, 0.5%). In this study, we found a new allele, KIR2DS4 * 016, with the difference in exon 5 comparing its most similar allele KIR2DS4 * 010. In the exon 5 of KIR2DS4 * 010, there is a 22bp-deletion, while the exon 5 of KIR2DS4 * 016 is normal. This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%. The sequence of the new allele sequence has been submitted to GenBank (accession no.: KC414890) and the IPD - KIR database (submission no.: IWS40001804), and was nominated by WHO nomenclature committee for HLA system. Conclusion:In this study, a sequence-based typing method for KIR2DS4 was established, and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.

OBJECTIVETo study the distribution of MICA alleles among ethnic Han Chinese blood donors from Shenzhen and their linkage disequilibrium with HLA-B gene.

METHODSFor 143 randomly selected blood donors, the MICA and HLA-B alleles were determined with a PCR-sequence based typing (SBT) method. Allelic frequency, haplotypic diversity and linkage disequilibrium were analyzed with a Pypop software.

RESULTSThirteen MICA and 35 HLA-B alleles were identified among the 143 blood donors, among which MICA*008:01 had the highest frequency (76/286), whilst MICA*008:01-HLA-B*40:01 and MICA*010-HLA-B*46:01 were the most common haplotypes. No novel allele was identified.

CONCLUSIONThe allele frequencies, haplotype diversities and linkage disequilibrium parameters under a high resolution can facilitate further studies and applications of the MICA and HLA-B genes.

OBJECTIVETo explore the association of KIR-HLA gene polymorphism with chronic myeloid leukemia (CML) among ethnic Hans from southern China.

METHODSA total of 172 adult CML patients and 480 unrelated healthy controls were screened for the presence of KIR with sequence-specific primers-PCR (PCR-SSP) and sequence-based typing (SBT) of HLA-A, -B and -C loci. Polymorphisms of the KIR-HLA system were analyzed at 4 levels, and the frequencies of KIR framework genes and KIR profiles, classⅠHLA ligands, matched KIR+HLA pairs and KIR-HLA compound profile were compared between the two groups. P values were calculated using SPSS 13.0 software.

RESULTSFor the CML group, the frequencies of HLA-C2 ligand, 2DL1+HLA-C2 pair and HLA-B Bw4-80I were significantly lower than those of the control group, suggesting a protective effect against CML (HLA-C2: OR=0.386, 95%CI:0.240-0.620, P<0.01; 2DL1+HLA-C2: OR=0.316, 95%CI:0.191-0.525, P<0.01; HLA-B Bw4-80I: OR=0.576, 95%CI:0.384-0.862, P<0.01). The frequencies of KIR2DL1 ligand (HLA-C2) and KIR3DL1 ligand (HLA-B Bw4-80I) in the CML group were significantly lower than that of the control group, suggesting that the HLA-C2 and HLA-B Bw4-80I expression is probably decreased in the CML patient group, which led to reduced inhibitory signal and enhanced activating signal of KIR2DL1and/or KIR3DL1NK cells. Notably, the frequency of KIR-HLA compound profiles ID2 (KIR AA1-HLA-C1/C1-Bw6/Bw6-A3/11) in CML patients significantly increased in the CML patient group compared with the control group, suggesting that the KIR-HLA compound profiles ID2 may be a risk factor for CML (OR=2.163, 95%CI 1.198-3.906, P<0.01).

CONCLUSIONAbove analysis has identified certain protective and risk factors for CML from the KIR-HLA system, which may provide a clue for the pathogenesis of leukemia and development of individualized immune therapy.

Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genetic Predisposition to Disease ; ethnology ; genetics ; Genotyping Techniques ; HLA Antigens ; genetics ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; ethnology ; genetics ; Odds Ratio ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Protein Isoforms ; genetics ; Receptors, KIR ; genetics ; Risk Factors

OBJECTIVETo study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.

METHODSGenomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.

RESULTSAmong all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.

CONCLUSIONThe present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; Haplotypes ; Humans ; Polymorphism, Genetic ; Receptors, KIR ; genetics ; Sequence Analysis, DNA ; methods

OBJECTIVETo study the genetic polymorphisms of human leukocyte antigen (HLA)- A, B, C, DRB1, DQA1, DQB1, DPA1and DPB1among ethnic Hans from southern China.

METHODS481 randomly selected individuals were genotyped using a polymerase chain reaction (PCR) sequence-based typing (SBT) method for the above genes. Their allele frequencies were determined by direct counting.

RESULTSIn total, 28 HLA-A, 57 HLA-B, 28 HLA-C, 40 HLA-DRB1, 18 HLA-DQA1, 17 HLA-DQB1, 6 HLA-DPA1and 21 HLA-DPB1alleles were identified. Among these, common alleles (with allelic frequencies > 0.05) included A*1101, A*2402, A*0207, A*3303, A*0201, B*40:01, B*46:01, B*58:01, B*13:01, B*15:02, C*01:02, C*07:02, C*03:04, C*03:02, C*08:01, C*03:03, C*04:01, DRB1*09:01, DRB1*15:01, DRB1*12:02, DRB1*08:03, DRB1*03:01, DRB1*04:05, DRB1*11:01, DQA1*01:02, DQA1*03:02, DQA1*03:03, DQA1*06:01, DQA1*01:03, DQA1*05:05, DQA1*01:04, DQA1*03:01, DQA1*05:01, DQB1*03:01, DQB1*03:03, DQB1*06:01, DQB1*05:02, DQB1*03:02, DQB1*02:01, DQB1*03:02, DQB1*06:02, DPA1*02:02, DPA1*01:03, DPA1*02:01, DPB1*05:01, DPB1*02:01, DPB1*13:01, DPB1*04:01and DPB1*02:02.For each of the locus, the overall frequencies of common alleles were 75.57%, 52.81%, 78.28%, 62.16%, 86.70%, 77.23%, 95.32% and 81.59%, respectively.

CONCLUSIONThe allelic frequencies of the 8 selected HLA loci among ethnic Hans from southern China may served as a reference for anthropology, legal medicine, transplantation and disease association studies.

Alleles ; Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; HLA-DP Antigens ; genetics ; HLA-DQ alpha-Chains ; genetics ; HLA-DQ beta-Chains ; genetics ; HLA-DRB1 Chains ; genetics ; Histocompatibility Antigens Class I ; genetics ; Histocompatibility Antigens Class II ; genetics ; Humans ; Linkage Disequilibrium ; Polymerase Chain Reaction ; Polymorphism, Genetic

BACKGROUND: Due to the polymorphism of HLA, a large number of ambiguities have been generated by conventional HLA typing techniques, and confirmed stereotypes of ambiguous results based on group-specific haploid full-length typing are rarely reported.OBJECTIVE: To analyze the accuracy of HLA-typing ambigulity based on group-specific haploid full-length sequencing. METHODS: The low-resolution results were used as the starting point for two ambiguous samples. Sanger sequencing (PCR-SBT) based on haploid full-length was performed after group-specific amplification. RESULTS AND CONCLUSION: One case showed a new A*02:03:01 allele, which was found a mutation in NT817 from C to T in comparison with A*11:01:01:01. The other case indicated another new C*07:02:01:01, which was found a mutation in NT879 from A to G in comparison with C*08:01:01. In conclusion, these results indicate that the group-specific haploid full-length sequencing method can be used to accurately classify HLA alleles and to discover new alleles.

BACKGROUND: Human leukocyte antigen-E (HLA-E) is one of non-classical HLA class I genes. Up to now, the polymorphism analysis is mainly aimed at the variation in exon 3 of HLA-E, which determines HLA-E*01:01 or HLA-E*01:03. However, the identification of the full-length HLA-E and its novel alleles is rare reported.OBJECTIVE: To establish the method of identification of HLA-E genomic full-length sequence, and to identify its novel alleles in healthy blood donors in Shenzhen, China.METHODS: Peripheral blood DNA samples were extracted from the subjects, and the amplified primers and sequencing primers in conserved regions were designed according to the sequences of HLA-E published in the IMGT/HLA database.A high-fidelity reaction system was used to amplify the genomic full-length of HLA-E, followed by sequencing,assembling, confirming and typing.RESULTS AND CONCLUSION: Herein, we successfully established the method for amplifying genomic full-length sequence and sequence-based typing. Two novel HLA-E alleles were nominated by WHO HLA Nomenclature committee as HLA-E*01:01:01:06 and HLA-E*01:01:01:07. Compared with the most related allele HLA-E*01:01:01:01,HLA-E*01:01:01:06 had one nucleotide change at nt-26(G->T) in 5'-promoter, and HLA-E*01:01:01:07 had one nucleotide change at nt3345(T->C) in 3'-UTR. The polymorphism data of genomic full-length HLA-E in Chinese individuals need to be filled, and the method we developed here supplies the key technique for the further studies.

Objective: To investigate the expression levels and clinical significance of serum suppressor of cytokine signaling-3 (SOCS3) and associated cytokines in HIV/TB co-infected patients. Methods: The serum levels of SOCS3, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17 and IL-22 were quantified by using enzyme-linked immunosorbent assays (ELISA) in 50 HIV-infected patients, 48 HIV/TB co-infected patients and 50 healthy donors. Pearson correlation analysis was used to analyze the correlation between SOCS3 and other seven cytokines. Results: Serum levels of SOCS3 expression in HIV/TB co-infection group were significantly higher than those in HIV-infection alone and the control group. There was also significant correlation between SOCS3 and IFN-γ, IL-4, IL-6, IL-10, IL-2 in HIV/TB co-infection group. Conclusions These findings indicated that SOCS3 may play an important role in the immune response of patients with HIV/TB co-infection and it may be helpful in the diagnosis of HIV/TB co-infection.