Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing
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DOI：
10.1016/j.ajhg.2016.06.023
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发表时间：
2016-08-04
影响因子：
9.8
通讯作者：
Parham, Peter
中科院分区：
文献类型：
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作者：
Norman, Paul J.;Hollenbach, Jill A.;Parham, Peter
自然杀伤（NK）细胞在人体免疫和生殖中的生理功能取决于杀伤细胞免疫球蛋白样受体（KIRs）与其HLA I类配体（HLA- a、HLA- b和HLA- c）之间的多种相互作用。包含KIR和HLA I类基因的基因组区域是不连接的，结构复杂，高度多态性。它们还与多种疾病密切相关，包括感染、自身免疫性疾病、癌症和妊娠障碍，以及移植和其他免疫疗法的疗效。为了促进这些特殊基因的研究，我们开发了一种方法，从基因组DNA中捕获，排序和分析13个KIR基因和HLA-A， HLA-B和HLA-C。我们还设计了一个生物信息学管道，将测序reads属性为特定的KIR基因，通过读取深度确定拷贝数，并为每个KIR基因调用高分辨率基因型。我们通过使用来自特征良好的细胞系的DNA，将其与已建立的HLA和KIR基因分型方法进行比较，并从1000个基因组序列数据中确定KIR基因分型，验证了该方法。这鉴定了116个以前未被鉴定的KIR等位基因，通过标准方法从源DNA测序证明这些等位基因都是真实的。仅对两个KIR基因的分析表明，1000个基因组个体中有22%具有以前未表征的等位基因或结构变异。我们所描述的方法适合于大规模分析，这些分析需要描述人群特征，并确定与疾病相关的精确HLA和KIR因素。该方法也适用于其他高多态性基因。
The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrumof diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.