KIR 3DL3基因测序分型中模棱两可结果的鉴定方法

Improved identification of ambiguous allele combinations observed in sequencing based typing of KIR3DL3 gene

目的探讨KIR3DL3基因测序分型中出现的模棱两可结果的鉴定方法。方法对南方汉族614例白血病患者的KIR3DL3基因的全部编码区进行测序分型,统计KIR3DL3模棱两可等位基因结果的种类及比例,针对每种模棱两可结果中等位基因编码区序列碱基的差异,设计组特异性PCR引物,进行组特异性KIR3DL3基因PCR扩增和再测序,鉴定KIR3DL3的基因型。结果共检出了7种新的KIR3DL3模棱两可的结果,其中3DL3*(00301,01002/00902,028)检出了7例,占1.14%(7/614);3DL3*(00102,01501/00601,00901/02102,023)、3DL3*(00402,01002/00802,028)、3DL3*(00402,01001/00801,028)分别检出了6例,均占0.98%(6/614);3DL3*(00301,01001/00901,028)、3DL3*(00301,00801/00402,00901)、3DL3*(00301,00802/00402,00902)分别检出了3例,均占0.49%(3/614)。这7种模棱两可组合分别采用组特异性PCR引物均能准确的鉴定出KIR3DL3基因型,其中3DL3*(00102,01501/00601,00901/02102,023)、3DL3*(00402,01001/00801,028)和3DL3*(00301,00802/00402,00902)的模棱两可组合样本均鉴定出1种基因型,其余的模棱两可组合均检出了2种不同的基因型。结论本文为鉴定KIR3DL3模棱两可的结果建立了可靠的鉴定方法,具有良好的实用价值。

Objective To better identify the KIR3 DL3 ambiguous allele combinations observed in sequencing-based typing（ SBT）. Methods A total of 614 cases of DNA samples from leukemia patients were subjected to sequence-based typing of KIR3 DL3 on a whole coding region scale. The number of each KIR3 DL3 ambiguity was calculated. Based on the nucleotide differences of alleles within each KIR3 DL3 ambiguous allele combination,group-specific PCR primers were designed to amplify the target allelic sequence. The ambiguities were re-identified by sequencing the amplified PCR products. Results Seven KIR3 DL3 ambiguous allele combinations were observed. The most common ambiguity was 3 DL3＊（ 00301,01002/＊00902,028） with a ratio of 1. 14%（ 7/614）; 3 DL3＊（ 00102,01501/00601,00901/02102,023）,3 DL3＊（ 00402,01002/00802,028） and 3 DL3＊（ 00402,01001/00801,028） were observed with ratios of 0. 98%（ 6/614） for each type; 3 DL3＊（ 00301,01001/00901,028）,3 DL3＊（ 00301,00801/00402,00901） and 3 DL3＊（ 00301,00802/00402,00902） were observed with a ratio of 0. 49%（ 3/614）,respectively. All those KIR3 DL3 ambiguous allele combinations could be distinguished by groupspecific PCR and sequencing retest. Only one KIR3 DL3 genotype was identified in 3 DL3＊（ 00102,01501/00601,00901/02102,023）,3 DL3＊（ 00402,01001/00801,028） and 3 DL3＊（ 00301,00802/00402,00902）,respectively; while the other ambiguity types yielded two different genotypes. Conclusion In this study,an efficient approach to identify the ambiguous allele combinations observed in KIR3 DL3 SBT has been established and promises a broad range of applications in KIR sequencing-based typing.