基于HLA组特异性单倍体全长测序方法对模棱两可结果的准确定型

Accurate determination of HLA ambiguous results based on group-specific haploid full-length sequencing

背景:传统的HLA测序分型技术都是对父系母系双倍体进行扩增,由于HLA多样性高,产生了大量的模棱两可难题,而采用基于组特异性单倍体全长测序分型方法来解决模棱两可结果的确认定型鲜有报道。目的:分析基于组特异性单倍体全长测序分型方法对HLA基因分型模棱两可结果准确定型的效果。方法:对2例模棱两可结果标本以低分辨率分型方法结果作为参考起点,通过组特异性扩增分离,基于等位基因全长单倍体的Sanger测序(PCR-SBT)分型。结果与结论:(1)2例模棱两可分型结果分别为A*02∶03∶01及C*07:02∶01∶01杂合新等位基因;(2)A*02∶03∶01杂合新等位基因与A*11∶01∶01∶01全长比较,在NT817由C发生突变为T;(3)C*07∶02∶01∶01杂合新等位基因与C*08∶01∶01比较,在NT879由A发生突变为G;(4)结果表明,以低分辨率分型方法结果作为参考起点,通过组特异性扩增分离,基于组特异性单倍体全长测序分型方法能准确定型HLA基因分型模棱两可结果并发现新等位基因。

BACKGROUND： Due to the polymorphism of HLA, a large number of ambiguities have been generated by conventional HLA typing techniques, and confirmed stereotypes of ambiguous results based on group-specific haploid full-length typing are rarely reported. OBJECTIVE： To analyze the accuracy of HLA-typing ambigulity based on group-specific haploid full-length sequencing. METHODS： The low-resolution results were used as the starting point for two ambiguous samples. Sanger sequencing（PCR-SBT） based on haploid full-length was performed after group-specific amplification. RESULTS AND CONCLUSION： One case showed a new A＊02：03：01 allele, which was found a mutation in NT817 from C to T in comparison with A＊11：01：01：01. The other case indicated another new C＊07：02：01：01, which was found a mutation in NT879 from A to G in comparison with C＊08：01：01. In conclusion, these results indicate that the group-specific haploid full-length sequencing method can be used to accurately classify HLA alleles and to discover new alleles.