HLA-B无效等位基因B*5408N的核苷酸序列分析

Identification and Sequence Analysis of A Null HLA-B Allele HLA-B*5408N Newly Detected

本研究探讨HLA新的等位基因HLA-B*5408N的分子基础。采用商用抽提试剂盒抽提样本DNA,利用单链特异性引物PCR方法扩增先证者HLA-B基因的第2-4外显子,对PCR扩增产物直接进行第2、3、4外显子双向测序分析。结果表明:先证者样本测序得到两个等位基因,其中一个等位基因为HLA-B*1527,另一个经blast验证其为新的等位基因,新的等位基因序列已递交GenBank(DQ295998,DQ295999,DQ296000)。与最接近的HLA-B*5401等位基因序列相比,新的等位基因仅在第3外显子上有1个核苷酸不同,即第553位G→T,这导致第161位氨基酸Glu(GAG)→终止密码(TAG),蛋白质的翻译提前终止。血清学方法未能检出HLA-B54抗原。结论:该等位基因为HLA-B无效表达等位基因,已被世界卫生组织HLA因子命名委员会正式命名为HLA-B*5408N。

The study was purposed to investigate the molecular genetic basis for HLA novel allele HLA-B ＊ 5408N in Chinese population. DNA was extracted from whole blood by commercial DNA extraction kit, the HLA-B exons 2 -4 of the proband was amplified by allele specific primers PCR and the amplified product was sequenced for exons 2,3 and 4 bidirectionally. The sequencing results showed HLA-B alleles of the proband as B ＊ 1527 and the novel allele. The sequences of the novel allele have been submitted to Genbank （DQ295998, DQ295999, DQ296000 ）. After blast analysis, the novel allele showed a single nucleotide mismatch with HLA-B ＊ 5401 in exon 3 at position 553 G→T, which resulted in an amino acid changing from Glu to premature stop codon at position 161. No the HLA-B54 antigen specificity expression in the proband cells was found using HLA-AB serological Typing Trays. It is concluded that this allele is a novel null allele and has been officially named B ＊ 5408N by the WHO Nomenclature Committee.