一例新的HLA-B等位基因B*5614的核苷酸序列分析

目的 研究HLA新的等位基因HLA-B*5614的分子基础。方法 样本DNA抽提采用盐析法，利用PCR方法扩增先证者HLA-B基因的第2～4外显子，PCR产物直接经TOPO转染克隆到质粒载体中分离其等位基因，对所得克隆进行第2～4外显子双向测序分析。应用序列特异性引物PCR方法证实测序所发现的突变。结果 先证者样本克隆测序得到两个等位基因，其中1个等位基因为B*1502，另一个经BLAST验证为新的等位基因，新的等位基因序列已递交GenBank(AY601726，AY601727，AY601728)。与最接近的B*5608等位基因序列相比，新的等位基因仅在第2外显子上有1个核苷酸不同，即第277位G→C，导致第93位氨基酸Cly→Arg。结论 该等位基因为新的HLA-B等位基因，被世界卫生组织HLA因子命名委员会正式命名为HLA-B*5614。

Identification and sequence analysis of a novel HLA-B*5614 allele

OBJECTIVE: To investigate the molecular genetics basis for a novel HLA allele, HLA-B*5614, in Chinese population. METHODS: DNA was extracted from whole blood by salting-out method. The HLA-B exons 2-4 of the proband was amplified and the amplified product was cloned using TOPO cloning sequencing kit to split the two alleles apart. Both strands of exons 2,3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. RESULTS: The sequencing results showed the HLA-B alleles of the proband as B*1502 and the novel allele. The sequences of the novel allele have been submitted to GenBank (AY601726, AY610727, AY610728). After BLAST analysis, the novel allele differs from B*5608 by a single nucleotide at position 277G-->C in exon 2. This results in an amino acid change from Gly to Arg at codon 93. CONCLUSION: This allele is a novel allele and has been officially named B*5614 by the WHO Nomenclature Committee.