HLA—A＊02：251新等位基因克隆测序鉴定及序列分析

Cloning and sequencing analysis of a novel allele HLA-A ＊ 02 ： 251

目的鉴定中国人群人类白细胞抗原（human leukocyte antigen, HLA）A＊02：251新等位基因，分析新等位基因遗传特征。方法采用聚合酶链反应一测序分型法（polymerase chain reaction-sequence based typing,PCR-SBT）对组织配型健康供、患者进行HLA基因分型，发现先证者核苷酸杂合序列与已知序列不匹配，不能指定先证者HLA等位基因型，对先证者DNA扩增HLA—A位点第2～4外显子，PCR产物经克隆到PMD18-T质粒载体中以获得单链核苷酸序列，对克隆所得产物进行HLA-A基因的第2～4外显子双向测序分析。结果发现先证者的一个HLA—A＊02：06：01基因被确认，而另一个HLAA基因为新等位基因，其序列被GenBank接受（编号为HM245348）。新等位基因序列通过IMGT／HLA数据库BLAST，与最相近的A＊02：01：01：01相比，在第3外显子上有1个核苷酸的不同，即第383位G〉C，密码子128GAG→GAC，氨基酸由谷氨酸（Glu）→天门冬氨酸（Asp）。供、患者HLA—A、B、C、DQB1位点等位基因不匹配。结论该等位基因为新的HLA—A＊02：251等位基因。中国人群HLA—A位点第3外显子核苷酸序列存在多态性。

Objective To identify a novel human leukocyte antigen （HLA） allele A ＊02：251 and analyze the sequences in Chinese population. Methods Routine HLA-A, B, -DRB1 high resolution genotyping for healthy Chinese donors and patients was performed with polymerase chain reaction-sequence based typing. An unknown HLA-A allele was initially detected by HLA typing in the healthy donor. Genomic DNA of the HLA-A locus in the proband was amplified, the amplified product was cloned by PMD18-T to split the two alleles, and selected clones were sequenced. Results The sequencing results showed that a normal A ＊ 02：06：01 and a novel A ＊ 02 ： 251 variant allele were identified. The sequence of the novel allele has been submitted to GenBank （HM245348）. Nucleotide sequence alignments with HLA-A allele from the IMGT/HLA Sequence Database showed that the novel A ＊02 variant allele differed from the closest allele A ＊02：01：01：01 by nt 383 G〉C （codon 128 GAG〉GAC） in exon 3, which resulted in one amino acid substitution of Glu〉Asp. The HLA-A, B, C and DQB1 alleles of the healthy donor did not match with that of the patient. Conclusion This novel allele is officially designated as HLA-A ＊02：251 by World Health Organization （WHO） Nomenclature Committee （Submission ID HWS10010755）. The sequence of HLA-A locus in exon 3 is confirmed to be polymorphic in Chinese population.