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Genotyping for human neutrophil antigen (HNA) systems is required in the investigation of disorders involving alloimmunisation to HNA. We established a polymerase chain reaction sequence-based typing method for genotyping HNA and determined the genotype and allele frequencies of HNA in the Zhejiang Han population of China.Four hundred, healthy unrelated Zhejiang Han individuals were recruited. Specific primers for HNA were designed and the polymerase chain reaction amplification conditions were optimised. Amplification amplicons were purified with enzyme digestion and then sequenced.The frequencies of the FCGR3B*01 and FCGR3B*02 alleles were 0.613 and 0.387; no FCGR3B*03 allele was found. The frequencies of the SLC44A2*1 and SLC44A2*2 alleles were 0.654 and 0.346, respectively, while the frequencies of the ITGAL*1 (HNA-5a) and ITGAL*2 (HNA-5b) alleles were 0.896 and 0.104. Only ITGAM*1 (HNA-4a) allele was found in this study. Six single nucleotide polymorphisms were confirmed on sequenced regions separate from HNA polymorphisms, including FCGR3B (IVS3+39GA and IVS3+52GA), CD177(172AG), SLC44A2 (IVS5-44AG and IVS7-15TC) and ITGAM (IVS3+118TC).The polymerase chain reaction sequence-based typing method for genotyping HNA is reliable. These data of HNA alleles frequencies could contribute to the analysis of alloimmunisation to HNA in China.

To identify two novel HLA alleles HLA-B*9536 and B*4612, in an individual.DNA was extracted from whole blood by Invitrogen DNA extraction kit. The amplification for HLA-B exons 2-4 of the proband was performed separately with allele group specific primers and the PCR products were directly sequenced for exons 2-4 in both direction.There were two novel HLA-B alleles in the proband. The sequences of the two alleles have been submitted to GenBank (EU081878 and EU081879). The two alleles have been officially named as B*9536 and B*4612 by the WHO Nomenclature Committee. The sequence of exons 2-4 of HLA-B*9536 showed one nucleotide difference in exon 3 at position 544 (G to A) comparing with the closest allele B*1505, which resulted in an amino acid change from Ala to Thr at codon 158. In the HLA-B*4612 allele, there was one nucleotide change in exon 3 at position 363 (G to A), when compared to the closest allele B*4601, which lead to an amino acid change from Arg to Ser at codon 97.Two novel HLA-B alleles were identified in one individual and have been officially named by the WHO Nomenclature Committee.