OBJECTIVE: Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes. METHODS: Three rare HLA-C alleles with zero mismatched PCR-SBT results were screened from clinical transplant matching samples, and the full-length sequence was detected by next-generation sequencing technology. RESULTS: The results of PCR-SBT typing of 3 samples were: HLA-C*03:04, 12:167; HLA-C*07:291, 15:02; HLA-C*01:43, 08:16. Other alleles were not in the CWD table of common and confirmed HLA alleles in China (version 2.3) except common allele HLA-C*03:04, HLA-C*15:02. NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles, and the three novel alleles had a base mutation in exons 6, 2, and 4, respectively. The novel allele sequences have been submitted to the Genbank database (MK629722, MK335474, MK641803), which were officially named HLA-C*03:04:74, HLA-C*15:192, HLA-C*08:01:25 by the WHO HLA Nomenclature Committee. The HLA high-resolution typing results of 3 samples were: HLA-C*03:04:74, HLA-C*12:03; HLA-C*07:02, HLA-C*15:192; HLA-C*01:02, HLA-C*08:01:25. CONCLUSION HLA typing results containing rare alleles should be treated cautiously, and the full-length sequence should be verified by NGS or cloning. The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing, which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.
Alleles ; Genotype ; HLA-C Antigens/genetics* ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Humans ; Polymerase Chain Reaction/methods* ; Sequence Analysis, DNA

OBJECTIVE: To verify a rare allele of human leukocyte antigen (HLA) and analyze its inheritance and 3D molecular structure. METHODS: PCR-sequence-based typing, PCR-single strand oligonucleotide polymorphism and single allele-specific sequencing were carried out to characterize the rare HLA-C allele and its transmission in the family. Its protein structure was modeled by using SWISS-MODEL, Phyre2 and FATCAT software. RESULTS: Analysis indicated that the rare allele (HLA-C*08:84) has transmitted from the proband's mother and has differed from HLA-C*08:01 by a single base (g.512G>C), resulting in substitution of an amino acid (p.Trp147Ser). Modeling of the 3D structure of the encoded protein indicated that the amino acid residue variation is located at the alpha 2 helix, which participates the formation of pocket F. Modeling of the structures of C*08:84, C*08:01, C*08:02, C*08:03 and C*08:22 has suggested significant variation in the peptide binding regions of the backbone, with root mean square errors being 1.70 nm, 1.79 nm, 0.71 nm and 1.70 nm, respectively. CONCLUSION A rare HLA-C*08:84 allele has been identified, and its clinical significance has been analyzed.
Alleles ; Base Sequence ; HLA-B Antigens/genetics* ; HLA-C Antigens/genetics* ; Humans ; Molecular Structure ; Sequence Analysis, DNA

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 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: The interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I regulates natural killer (NK) cell cytotoxicity and function. The impact of NK cell alloreactivity through KIR in liver transplantation remains unelucidated. Since the frequency of HLA-C and KIR genotypes show ethnic differences, we assessed the impact of HLA-C, KIR genotype, or KIR-ligand mismatch on the allograft outcome of Korean liver allografts. METHODS: One hundred eighty-two living donor liver transplant patients were studied. Thirty-five patients (19.2%) had biopsy-confirmed acute rejection (AR), and eighteen (9.9%) had graft failure. The HLA-C compatibility, KIR genotypes, ligand-ligand, and KIR-ligand matching was retrospectively investigated for association with allograft outcomes. RESULTS: Homozygous C1 ligands were predominant in both patients and donors, and frequency of the HLA-C2 allele in Koreans was lower than that in other ethnic groups. Despite the significantly lower frequency of the HLA-C2 genotype in Koreans, donors with at least one HLA-C2 allele showed higher rates of AR than donors with no HLA-C2 alleles (29.2% vs 15.7%, P=0.0423). Although KIR genotypes also showed ethnic differences, KIR genotypes and the number of activating KIR/inhibitory KIR were not associated with the allograft outcome. KIR-ligand mismatch was expected in 31.6% of Korean liver transplants and had no impact on AR or graft survival. CONCLUSIONS: This study could not confirm the clinical impact of KIR genotypes and KIR-ligand mismatch. However, we demonstrated that the presence of HLA-C2 allele in the donor influenced AR of Korean liver allografts.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Female ; Genotype ; Graft Rejection ; Graft Survival ; HLA-C Antigens/*genetics ; Homozygote ; Humans ; Killer Cells, Natural/cytology/immunology ; Ligands ; *Liver Transplantation ; Male ; Middle Aged ; Proportional Hazards Models ; Receptors, KIR/chemistry/*genetics/metabolism ; Republic of Korea ; Tissue Donors ; Transplantation, Homologous

OBJECTIVETo investigate the frequency of HLA-Cw and its KIR2D genotypes in Han blood donor population in Chinese Nanjing area and to analyze the match and distinguish modes of them so as to provide the basis for further studying their roles in incidence and development of disease.

METHODSThe PCR-SSP was used to genotyping of HLA-Cw and KIR2D for 241 Han blood donors in Jiangsu Provincial blood center; according to distingush modes of HLA-Cw and KIR2D genes, the distingushed results of HLA-Cw and corresponding activating or inhibitory KIR2D receptors of individuals were analyzed.

RESULTSThe frequency of HLA-C1 expression in donor population of Nanjing area was 76.35% which was much higher than that of HLA-C2 expression (23.65%); the expression C1/C2 alleles was accorded with Handy-weinberg balance. The expression frequencies of 5 KIR2Ds (L1, L2, L3, S1 and S2) matched to HLA-Cw were 97.93%, 29.05%, 98.34%, 29.05% and 21.16%, respectively. The match of HLA-C1/C2 to 2DL1⁺/2DL2⁻/2DL3⁺/2DS1⁻/2DS2⁻ was predominated (75/241). Couclusion: The polymorphism data of HLA-Cw and 5 KIR2Ds from blood donors in Chinese Nanjing area has been provided in this study. The match analysis found that the expression of inhibitory HLA-Cw-KIR is higher than that of activated HLA-Cw-KIR, suggesting that the HLA-Cw/KIR2D combination is characterized by preponderance of inhibitory signal pathway.

Alleles ; Asian Continental Ancestry Group ; genetics ; Blood Donors ; China ; Ethnic Groups ; genetics ; Genotype ; HLA-C Antigens ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Receptors, KIR ; genetics

The aim of this study was to investigate the relatively frequencies of alleles in the HLA-C*04:01:01G group and to analyze their relations with HLA-A and -B loci. DNA samples previously typed as HLA-C*04:01:01G were sequentially selected. The sequences for exon 2 to 7 of the HLA-C locus were analyzed by polymerase chain reaction sequence-based typing(PCR-SBT). The HLA-A, -B, -DRB1 and -DQB1 loci were genotyped using PCR-SBT method. The results showed that 178 samples (94.2%) and 11 samples (5.8%) were assigned as HLA-C*04:01:01 and HLA-C*04:82 respectively among 189 samples previously typed as HLA-C*04:01:01G. 72 haplotypes associated with HLA-C*04:01:01 and C*04:82 were found, in which the frequencies of 26 haplotypes were over 0.0050. HLA-C*04:01:01 was strongly related with A*02:03, A*02:07, A*11:01, A*33:03, B*13:01, B*15:01, B*15:05, B*15:27, B*40:01, B*54:01 alleles, while HLA-C*04:82 was related with B*40:01. It is concluded that HLA-C*04:01:01 and HLA-C*04:82 alleles were confirmed in the HLA-C*04:01:01G group, which should be discriminated by the routine HLA genotyping.
Alleles ; Base Sequence ; Gene Frequency ; Genotype ; Genotyping Techniques ; HLA-C Antigens ; genetics ; Haplotypes ; Humans

OBJECTIVETo investigate the genetic basis for a novel allele HLA-C*01:78.

METHODSGenomic DNA was extracted from peripheral blood using a QIAGEN quick DNA extraction kit. The regions encompassing HLA-C from exon 1 to intron 3 and intron 3 to 3'UTR were amplified and cloned using a cloning sequencing kit in order to split the two alleles apart. Selected clones were sequenced to include exons 2 to 4.

RESULTSSequencing results have indicated the HLA-C alleles of the proband to be a novel C*03:04 allele. The sequence has been submitted to GenBank (KF049216). BLAST analysis has confirmed the novel allele to have one nucleotide difference as C*01:03 at genomic nt316C>A (codon 82CGC>AGC) in exon 2, which has resulted in replacement of one amino acid (82R>S).

CONCLUSIONThe novel allele has been officially named as C*01:78 by the WHO Nomenclature Committee. The HLA allele type of the proband was therefore A*02:07, 24:02; B*40:01, 46:01; C*01:78, 03:04; DQB1*05:02, 05:02; DRB1*16:02, 16:02.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Exons ; Female ; HLA-C Antigens ; genetics ; Humans ; Introns ; Leukemia ; genetics ; Male ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo develop a method for separating the human leukocyte antigen (HLA)-A, -B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results.

METHODSBased on sequence information of HLA alleles from the IMGT/HLA database, the 5-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis.

RESULTSAmong the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples.

CONCLUSIONThe developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.

Genotype ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; Haploidy ; Humans ; Molecular Probe Techniques ; instrumentation ; Polymerase Chain Reaction ; instrumentation ; methods ; Streptavidin ; chemistry

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
Adult ; Amino Acid Sequence ; Animals ; Binding Sites ; Epitopes ; chemistry ; immunology ; metabolism ; Female ; Genotype ; HEK293 Cells ; HLA-A2 Antigen ; metabolism ; Hepatitis B Core Antigens ; chemistry ; immunology ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Hydrogen Bonding ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Middle Aged ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism