Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

Sequence analysis of a novel HLA-A?01 ∶130 allele and modeling of three-dimensional structure of HLA molecule / 中国免疫学杂志

Objective:To confirm the novel allele HLA-A*01∶130 and analyzed the nucleotide sequence of the abnormal reaction pattern.Methods: The HLA typing of sample DNA was performed by PCR-SBT.The ambiguous novel HLA allele was confirmed with single stranded SBT method,then DNA sequencing was performed to identify the difference between the novel allele and HLA-A?01:66 allele.Finally, it was modeled by Swiss-Model to three-dimensional structure of HLA Molecule.Results: The novel allele was not the same with all known HLA-A allele sequence.After analysis,there was one nucleotide differed from the A?01:66 at position 368 where A→G( codon 99 TAT→TGT) resulting in a coding change,99 Tyr was changed to Cys.The amino acid substitution at residue 99 the HLA polypeptide was located in a beta-sheet of antigenic peptide-binding region.Conclusion: The allele is a novel allele that has now been officially named as HLA-A*01∶130 by the World Health Organization( WHO) HLA Nomenclature Committee.

Identification of a novel human leukocyte antigen allele, HLA-A * 31 ∶ 22 / 中华微生物学和免疫学杂志

Objective To identify and confirm a novel HLA allele.Methods A new human leukocyte antigen A allele was found during routine HLA genotyping by polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSOP) and sequencing-based typing (SBT).HLA-A locus was amplified from exon 1 through exon 8,and the nucleotide sequence of exon 2 to exon 4 for HLA-A were sequenced in both directions.Results The novel HLA-A * 31 allele is identical to A * 31 ∶ 01 ∶ 02 with an exception of one base substitution at position 245 of exon 2 where an ' A' change to ' C' resulting in codon 82 changed from GAG to GCG.Conclusion A novel HLA allele,A * 31 ∶ 22,was identified,and was named officially by the WHO Nomenclature Committee for factors of the HLA system.

A novel HLA allele, HLA-B * 40:74, detected in a Chinese individual / 中华微生物学和免疫学杂志

ObjectiveTo identify and confirm a novel HLA allele in a Chinese individual.MethodsA new HLA allele was found during routine HLA genotyping by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) and sequencing-based typing (SBT).ResultsThe new sequence differs from HLA-B * 40:01:01 by two nucleotide substitutions in exon 2 at positions 103 (G＞T) and 106 (A＞G) ; These mutations result in two codon changes:at codon 35 (GCC＞TCC) where an alanine (A) is substituted by a serine(S) and at codon 36(ATG＞GTG) where a methionine(M) is substituted by a valine (V).ConclusionA novel HLA allele,HLA-B * 40:74,was identified,and was named officially by the WHO Nomenclature Committee (HWS10004518 - EF458488).

Single-Tube Polymerase Chain Reaction Amplification Protocol for Sequencing-Based Typing of Human Leukocyte Antigen DRB1 / 中国普外基础与临床杂志

Objective To develop a new single-tube polymerase chain reaction amplification (ST Amp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen DRB1(HLA-DRB1).Methods A set of 7 group-specific exonic 5′ amplification primers and a single generic 3′ primer were included together in a single PCR mix to facilitate a single PCR amplification per sample for HLA-DRB1 typing.Results All samples were successfully typed, the typing result was accurate and repeatable.Conclusion ST Amp technique has resulted in the ability to perform high-resolution, high-specificity and high-throughput HLA-DRB1 typing by DNA sequencing.

Identification of a novel HLA-A~*0290 allele by sequencing-based typing / 中国输血杂志

G in exon 2. This results in an amino acid change from His to Asp at codon 74. Conclusion The novel allele has been confirmed as a new HLA allele and it is officially named HLA-A*0290 by WHO Nomenclature Committee for Factors of the HLA System in Oct. 2005.