OBJECTIVETo establish a polymerase chain reaction sequencing-based typing (PCR SBT) method for HLA-DPB1 exons 2 and 3, and to analyze their polymorphisms.

METHODSBased on the sequences of HLA-DPB1 loci, locus-specific primers were designed and applied to amplify the target sequences encompassing the entire exons 2 and 3 of HLA-DPB1. The amplification products were digested by enzymes and directly sequenced in both directions. The genotype was assigned by Assign 3.5+ SBT software.

RESULTSSpecific target fragment was obtained with the PCR amplification, and good quality electropherogram was derived by direct sequencing. Among 242 individuals from Zhejiang Han population, 18 HLA-DPB1 alleles were detected. Alleles with a frequency of > 0.05 have included DPB1*05:01:01/135:01 (0.4112), DPB1*02:01:02 (0.1901), DPB1*04:01:01 (0.1136) and DPB1*02:02 (0.0620). A novel HLA-DPB1*168:01 allele has also been identified. Nine polymorphism sites were founded in the exon 3 region, which included a new SNP site 517 A>T.

CONCLUSIONThe PCR-SBT method for exons 2 and 3 of HLA-DPB1 is reliable, which allowed detection of polymorphisms in exon 3 of the HLA-DPB1 gene.

Alleles ; Exons ; HLA-DP beta-Chains ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic

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

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

OBJECTIVE： To evaluate clinical efficacy of Guanxin shutong capsule （GSC） in the adjuvant treatment of unstable angina pectoris （UAP）， and to provide evidence-based reference for clinical treatment of UAP. METHODS： Retrieved from PubMed， Embase， Cochrane library， CBM， CNKI， VIP and Wanfang database， randomized controlled trials（RCTs）about routine treatment （trial group） of GSC combined with western medicine versus western medicine routine treatment （control group） in the treatment of UAP were collected during database establishment to Oct. 11th ， 2018. After data extraction of included literatures and quality evaluation with Cochrane bias risk evaluation tool 5.1.0， Meta-analysis of total response rate of angina pectoris， total response rate of ECG， blood lipid levels （TC， HDL-C， LDL-C， TG）， the level of hs-CRP were performed by using Rev Man 5.2 statistical software. TSA 0.9 software was used for trial sequential analysis （TSA） of the total response rate of angina pectoris and response rate of ECG. RESULTS： A total of 11 RCTs were included， involving 946 patients. Results of Meta-analysis showed that total response rate of angina pectoris [RR=1.24，95％CI（1.16，1.32），P＜0.001] and total response rate of ECG [RR＝1.22，95％CI（1.11，1.34），P＜0.001] in trial group were significantly higher than control group. The improvement of TC [SMD＝－1.55，95％CI（－1.81，－1.29），P＜0.001]， TG [SMD＝－0.84，95％CI（－1.08，－0.60），P＜0.001]， HDL-C [SMD＝0.15，95％CI（0.06，0.25），P＝0.001]， LDL-C [SMD＝－0.62，95％CI（－0.76，－0.48），P＜0.001] and hs-CRP [SMD＝－2.54，95％CI（－3.88，－1.88），P＜0.001] in trial group were better than control group. TSA analysis showed that the evidence of Meta-analysis was reliable. CONCLUSIONS： GSC combined with western medicine routine treatment can improve total response rate of angina pectoris， total response rate of ECG， blood lipid and hs-CRP level of UAP patients.