南方汉族人群功能性KIR框架基因测序分型中模棱两可的结果及解决策略

A strategy to clarify ambiguities during genotyping of functional KIR framework genes by sequencing-based typing among ethnic Hans from southern China

目的探讨南方汉族人群中功能性KIR框架基因全部编码区测序分型中模棱两可结果的种类及其解决方法。方法对306份南方汉族人群的14个功能性KIR框架基因的全部编码区进行测序分型，统计模棱两可结果的种类及比例，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及测序引物，进行组特异性KIR等位基因PCR扩增和再测序，以鉴定等位基因型别。结果发现KIRZDL5、3DL2和3DL3存在6种模棱两可的结果，种类及其比例依次为：3D阳＊（002，007／010，015）占12．09％（37／306），（2DLSA＊001，2DLSB＊006／2DL5A＊012、2DLSB＊008）占5．88％（18／306），3DL3＊（001，010／009，048）占3．59％（11／306），3DL2＊（007，008／016，027）占2．29o．4（7／306），3DL3＊（00801，048／01001，026）及3DL3＊（00802，048／01002，026）均占1．31％（4／306）。除了（2DL5A＊001，2DL5B＊006／2DLSA＊012，2DL5B＊008）、3DL2＊（007，008／016，027）两组模棱两可结果均分别只检出其中的一种基因型外，其余的4组模棱两可结果每组中均检出了不同的基因型。结论我们建立了有效的组特异性引物扩增和测序方法鉴定模棱两可的结果，在KIR测序分型中具有广泛的应用价值。

Objective To categorize ambiguous allelic combinations encountered in genotyping of functional KIR genes by sequencing-based typing of the entire coding sequence and develop an efficient approach to identify such ambiguities. Methods Fourteen KIR functional framework genes from 306 ethnic Chinese individuals were genotyped for the entire encoding sequence. The number of ambiguities was directly counted. Based on the differences within each ambiguous allelic combination, group-specific PCR primers and sequencing primers for amplifying the target allelic sequence were designed. The PCR products were then subjected to sequencing in order to identify the ambiguities. Results The 14 functional KIR genes were subjected to sequencing-based typing （SBT） for the entire encoding sequence. Six ambiguous allelic combinations were identified. The most common ambiguity 3DL2 ＊ （002, 007/010, 015） has accounted for 12.09% of the 306 tested samples. The remaining 5 ambiguities were （2DL5A ＊ 001, 2DL5B O06/2DL5A ＊ 012, 2DLSB ＊ 008）, 3DL3 ＊ （001, 010/009, 048）, 3DL2 ＊ （007, 008/016, 027）, 3DL3 （00801, 048/01001, 026）and 3DL3 ＊ （00802, 048/01002, 026）have accounted for 5. 88% （18/306）, 3.59N （11/306）, 2.29N （7/306）, 1.31N （4/306） and 1.31N （4/306） of all samples, respectively. For two ambiguities （2DLSA ＊ 001, 2DL5B ＊ 006/2DL5A ＊ 012, 2DL5B ＊ 008） and 3DL2 ＊ （007, 008/016, 027） subjected to group-specific PCR and re-sequencing, only one demonstrable genotype was identified, While for in each of other four ambiguities subjected to group-specific PCR and re-sequencing, two different genotypes were identified. Conclusion An efficient approach by group-specific PCR and sequencing retest has been established to clarify the ambiguities during SBT testing for functional KIR framework genes, which may have a broad application in KIR sequencing-based typing.