title: A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China.
abstract: HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allelic distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers (PCR-SSP) to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles (> 99% allele frequency) reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A*0209 and HLA-A*0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis (FCM) with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A*0201 ranks the first (allele frequency = 15.5%), followed by A*0207 (5.8%), A*0206 (4.7%), A*0203 (2.6%), A*0210 (0.7%), and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.
authors.affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Liang
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhu
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Liang
authors.forename: Xiufang
authors.initials: X
authors.lastname: Weng
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Lu
authors.forename: Cai'e
authors.initials: C
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Xiongwen
authors.initials: X
authors.lastname: Wu
mesh_terms: D002681:China; D015789:HLA-A2 Antigen / Q000145:classification* / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction
chemical_list: D015789:HLA-A2 Antigen